Cytotoxicity of ZnO NPs towards fresh water algae Scenedesmus obliquus at low exposure concentrations in UV-C, visible and dark conditions.

Continuous increase in the usage of ZnO nanoparticles in commercial products has exacerbated the risk of release of these particles into the aquatic environment with possible harmful effects on the biota. In the current study, cytotoxic effects of two types of ZnO nanoparticles, having different initial effective diameters in filtered and sterilized lake water medium [487.5±2.55 nm for ZnO-1 NPs and 616.2±38.5 nm for ZnO-2 NPs] were evaluated towards a dominant freshwater algal isolate Scenedesmus obliquus in UV-C, visible and dark conditions at three exposure concentrations: 0.25, 0.5 and 1 mg/L. The toxic effects were found to be strongly dependent on the initial hydrodynamic particle size in the medium, the exposure concentrations and the irradiation conditions. The loss in viability, LDH release and ROS generation were significantly enhanced in the case of the smaller sized ZnO-1 NPs than in the case of ZnO-2 NPs under comparable test conditions. The toxicity of both types of ZnO NPs was considerably elevated under UV-C irradiation in comparison to that in dark and visible light conditions, the effects being more enhanced in case of ZnO-1 NPs. The size dependent dissolution of the ZnO NPs in the test medium and possible toxicity due to the released Zn(2+) ions was also noted. The surface adsorption of the nanoparticles was substantiated by scanning electron microscopy. The internalization/uptake of the NPs by the algal cells was confirmed by fluorescence microscopy, transmission electron microscopy, and elemental analyses.

Long-term regulation of carotid body function: acclimatization and adaptation--invited article.

Physiological responses to hypoxia either continuous (CH) or intermittent (IH) depend on the O(2)-sensing ability of the peripheral arterial chemoreceptors, especially the carotid bodies, and the ensuing reflexes play important roles in maintaining homeostasis. The purpose of this article is to summarize the effects of CH and IH on carotid body function and the underlying mechanisms. CH increases baseline carotid body activity and sensitizes the response to acute hypoxia. These effects are associated with hyperplasia of glomus cells and neovascularization. Enhanced hypoxic sensitivity is due to alterations in ion current densities as well as changes in neurotransmitter dynamics and recruitment of additional neuromodulators (endothelin-1, ET-1) in glomus cells. Morphological alterations are in part due to up-regulation of growth factors (e.g. VEGF). Hypoxia-inducible factor-1 (HIF-1), a transcriptional activator might underlie the remodeling of carotid body structure and function by CH. Chronic IH, on the other hand, is associated with recurrent apneas in adults and premature infants. Two major effects of chronic IH on the adult carotid body are sensitization of the hypoxic sensory response and long-lasting increase in baseline activity i.e., sensory long-term facilitation (LTF) which involve reactive oxygen species (ROS) and HIF-1. In neonates, chronic IH leads to sensitization of the hypoxic response but does not induce sensory LTF. Chronic IH-induced sensitization of the carotid body response to hypoxia increases the likelihood of unstable breathing perpetuating in more number of apneas, whereas sensory LTF may contribute to increased sympathetic tone and systemic hypertension associated with recurrent apneas.

NADPH oxidase is required for the sensory plasticity of the carotid body by chronic intermittent hypoxia.

Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a approximately 12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT(2) receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H(2)O(2) generated from O(2).(-) contributes to sLTF. Priming with H(2)O(2) elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Modulation of the hypoxic sensory response of the carotid body by 5-hydroxytryptamine: role of the 5-HT2 receptor.

Previous studies have shown that glomus cells of the carotid body express 5-hydroxytryptamine (5-HT). The aim of this study was to elucidate the role of 5-HT on the hypoxic sensory response (HSR) of the carotid body. Sensory activity was recorded from multi-fiber (n=16) and single-fiber (n=8) preparations of ex vivo carotid bodies harvested from anesthetized, adult rats. 5-HT (3 microM) had no significant effect on the magnitude or on the onset of the HSR. However, 5-HT consistently prolonged the time necessary for the sensory activity to return to baseline following the termination of the hypoxic challenge. Ketanserin (40 microM), a 5-HT2 receptor antagonist completely prevented 5-HT-induced prolongation of the HSR, whereas had no effect on the control HSR (onset, magnitude, and time for decay without 5-HT). Carotid bodies expressed 5-HT, but hypoxia did not facilitate 5-HT release. These observations suggest that 5-HT is not critical for the HSR of the rat carotid body, but it modulates the dynamics of the HSR via its action on 5-HT2 receptors.

Release of substance P by low oxygen in the rabbit carotid body: evidence for the involvement of calcium channels.

Carotid bodies from diverse species contain substance P (SP), an 11-residue peptide that belongs to the tachykinin peptide family. Previous studies indicated that SP is excitatory to the carotid body and is associated with sensory response to hypoxia. However, release of SP from the carotid body during hypoxia has not been documented. In the present study, we determined whether hypoxia releases SP from the carotid body and further characterized the mechanism(s) associated with SP release by low oxygen. The release of SP from superfused rabbit carotid body was determined by an enzyme immunoassay (EIA). SP-like immunoreactivity was localized to many glomus cells and nerve fibers and the concentration of SP in the rabbit carotid body was 1.5+/-0.1 ng/mg protein. For release studies, carotid bodies (n=56) were superfused with a modified Tyrode medium containing Hepes buffer, pH 7.4, saturated with either room air (normoxia) or hypoxic gas mixtures. The basal release of SP during normoxia was 51.0+/-1.5 fmol/min per mg protein. Hypoxia increased SP release from the carotid body and the magnitude of release is dependent on the severity of hypoxic stimulus. Moderate hypoxia (pO2, 79+/-4 mmHg) stimulated SP release by approximately 50%, whereas SP release during severe hypoxia (pO2, 11+/-6 mmHg) was 2-fold higher than the normoxic control. A similar pattern of SP release was also observed when superfusion medium containing CO2-HCO3 buffer, pH 7.4, was used for release studies. To examine the mechanism(s) associated with hypoxia-induced SP release from the carotid body, moderate level of hypoxia (12% O2+N2) was used. Omission of calcium in the superfusion medium markedly attenuated hypoxia-induced SP release (>95%), whereas the basal release of SP was unaffected. Cd2+ (100 microM), a voltage-dependent Ca2+ channel blocker, abolished hypoxia-induced SP release. About 85% of SP release by hypoxia was inhibited by omega-conotoxin GVIA (1 microM), an N-type Ca2+ channel blocker, whereas nitrendipine (1.5 microM), an inhibitor of L-type Ca2+ channel partially attenuated ( approximately 65%) hypoxia-induced SP release. By contrast, omega-agatoxin TK (50 nM), a P/Q-type Ca2+ channel inhibitor, had no significant effect (P>0.05, n=6). These results suggest that SP is released from the rabbit carotid body by hypoxia that depends on the severity of the hypoxic stimulus. Further, SP release by hypoxia is a calcium-dependent process and is primarily mediated by N- and L-type Ca2+ channels.

A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Interaction between catecholamines and neuropeptides in the carotid body: evidence for dopamine modulation of neutral endopeptidase activity.

Carotid body (CB) contains multiple neurochemicals that include catecholamines (CA) and neuropeptides. They are involved in the modulation of sensory response of the carotid body. Based on observations from the central nervous system, we hypothesized that CA modulates neuropeptide metabolism in CB. To test our hypothesis, fetal calf carotid body model was used. Immunocytochemical analysis showed that fetal calf carotid body expresses both tyrosine hydroxylase, and neutral endopeptidase-like immunoreactivity. To assess the effect of CA, thin slices of fetal calf carotid body were incubated with 50-500 microM of dopamine (DA) at 37 degrees C for 1 hour. As an index of neuropeptide metabolism, the activity of neutral endopeptidase (NEP), a major degrading enzyme of neuropeptides in CB was determined in the membrane-enriched and soluble fractions of the carotid body. CBs incubated with medium lacking DA served as control. On average, NEP activities of the membrane-enriched, and soluble fractions of the untreated CB were 4.8 +/- 0.2 and 6.7 +/- 0.2 pmole per hour per mg of CB respectively. At concentrations less than 200 microM, DA enhanced NEP activity of the membrane fraction (approximately 60%) whereas inhibition of NEP was observed in the soluble fraction (approximately 62%). At concentrations > 200 microM, DA inhibited NEP activity of the two fractions. When CBs were incubated with DA in the presence of sodium dithionite, an oxygen scavenger, DA, even at higher concentrations, stimulated NEP activity of the membrane-enriched fraction. The above results demonstrate that DA modulates neuropeptide metabolism in CB via a non-receptor-mediated mechanism involving a direct interaction with NEP.

Role of substance P in neutral endopeptidase modulation of hypoxic response of the carotid body.

Carotid body expresses neutral endopeptidase (NEP)-like enzyme activity and phosphoramidon, an inhibitor of NEP augments sensory response of the carotid body to hypoxia (Kumar et al., 1990). NEP hydrolyzes substance P (SP) and methionine enkephalin (Met-ENK) in the nervous system. In the present study, we determined whether NEP hydrolyzes Met-ENK and SP in the carotid body and whether these peptides contribute to the phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anaesthetized adult cats. HPLC analysis showed that both SP and Met-ENK were hydrolyzed by the carotid body. Phosphoramidon (400 microM) markedly inhibited SP (approximately 90%) but had only marginal effect on Met-ENK hydrolysis (approximately 15%). Sensory responses of the carotid body in vitro to hypoxia (pO2, 68 +/- 6 mmHg) and SP (10 nmoles) were potentiated by phosphoramidon by approximately 80% and approximately 275% respectively (p < 0.01). SP-receptor antagonist abolished phosphoramidon-induced potentiation of the sensory response to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and SP plays a major role in the potentiation of the hypoxic response of the carotid body by phosphoramidon.

Involvement of substance P in neutral endopeptidase modulation of carotid body sensory responses to hypoxia.

Previously, we showed that carotid bodies express neutral endopeptidase (NEP)-like enzyme activity and that phosphoramidon, a potent inhibitor of NEP, potentiates the chemosensory response of the carotid body to hypoxia in vivo. NEP has been shown to hydrolyze methionine enkephalin (Met-Enk) and substance P (SP) in neuronal tissues. The purpose of the present study is to determine whether NEP hydrolyzes Met-Enk and SP in the carotid body and if so whether these peptides contribute to phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anesthetized adult cats (n = 72 carotid bodies). The hydrolysis of Met-Enk and SP was analyzed by HPLC. The results showed that both SP and Met-Enk were hydrolyzed by the carotid body, but the rate of Met-Enk hydrolysis was approximately fourfold higher than that of SP. Phosphoramidon (400 microM) markedly inhibited SP hydrolysis ( approximately 90%) but had only a marginal effect on Met-Enk hydrolysis ( approximately 15% inhibition). Hypoxia (PO(2), 68 +/- 6 Torr) as well as exogenous administration of SP (10 and 20 nmol) increased the sensory discharge of the carotid body in vitro. Sensory responses to hypoxia and SP (10 nmol) were potentiated by approximately 80 and approximately 275%, respectively (P < 0.01), in the presence of phosphoramidon. SP-receptor antagonists Spantide (peptidyl) and CP-96345 (nonpeptidyl) either abolished or markedly attenuated the phosphoramidon-induced potentiation of the sensory response of the carotid body to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and that SP is involved in the potentiation of the hypoxic response of the carotid body by phosphoramidon.

Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea.

Identification of common dietary substances capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. An antioxidant-rich polyphenolic fraction isolated from green tea (green tea polyphenols, GTPs) has been shown to possess anti-inflammatory and anticarcinogenic properties in experimental animals. In this study we determined the effect of oral consumption of GTP on collagen-induced arthritis in mice. In three independent experiments mice given GTP in water exhibited significantly reduced incidence of arthritis (33% to 50%) as compared with mice not given GTP in water (84% to 100%). The arthritis index also was significantly lower in GTP-fed animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-gamma, and tumor necrosis factor alpha in arthritic joints of GTP-fed mice. Histologic and immunohistochemical analysis of the arthritic joints in GTP-fed mice demonstrated only marginal joint infiltration by IFN-gamma and tumor necrosis factor alpha-producing cells as opposed to massive cellular infiltration and fully developed pannus in arthritic joints of non-GTP-fed mice. The neutral endopeptidase activity was approximately 7-fold higher in arthritic joints of non-GTP-fed mice in comparison to nonarthritic joints of unimmunized mice whereas it was only 2-fold higher in the arthritic joints of GTP-fed mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of GTP-fed mice. Taken together our studies suggest that a polyphenolic fraction from green tea that is rich in antioxidants may be useful in the prevention of onset and severity of arthritis.

When is facial pain trigeminal neuralgia?

The pain of trigeminal neuralgia can be excruciating and debilitating. Fortunately, effective medical and surgical therapies for the disorder exist. Successful treatment hinges on thorough history taking and accurate diagnosis. Diagnostic evaluation of patients with orofacial pain should include complete head and neck, dental, and neurologic examinations combined with radiologic imaging of the head and appropriate laboratory tests.

Release of dopamine and norepinephrine by hypoxia from PC-12 cells.

We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (PO2 of medium approximately 40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of PO2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was approximately 11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 microM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in PO2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

Peptidases of the peripheral chemoreceptors: biochemical, immunological, in vitro hydrolytic studies and electron microscopic analysis of neutral endopeptidase-like activity of the carotid body.

The purposes of the present study are to identify and characterize the major peptidase(s) that may be involved in the inactivation of neuropeptides in the mammalian carotid body. Measurements of a number of peptidase activities in the cell-free extract of the cat carotid body using specific substrates and inhibitors indicated that the previously identified neutral endopeptidase (NEP)-like activity [Kumar et al., Brain Res., 517 (1990) 341-343] is the major peptidase in the chemoreceptor tissue. The NEP-like activity of the carotid body was further characterized using a monoclonal antibody to human neutral endopeptidase, EC 3.4.24.11. Immune blot analysis indicated strong immunoreactivity toward the cat and calf carotid bodies but a weak cross-reactivity with the rabbit carotid body. Furthermore, western blot analysis of the cat carotid body extract revealed the presence of a major 97-kDa protein and a minor 200-kDa protein. The 97-kDa NEP form of the carotid body was comparable to EC 3.4.24.11 and was consistent with its reported molecular weight suggesting NEP-like activity of the carotid body is structurally similar to the neutral endopeptidase, EC 3.4.24.11. In order to assess whether NEP is the primary peptide degrading activity in the cat carotid body in vitro hydrolysis studies using substance P (SP) as a model peptide were performed. HPLC analysis showed that SP is hydrolyzed maximally at pH 7.0 by carotid body peptidases with the formation of SP(1-7) and SP(1-8) as stable intermediates. Inhibitors specific to NEP also inhibited the SP-hydrolyzing activity of the carotid body. Analyses of the cell-free extracts showed the occurrence of both NEP and SP-hydrolyzing activities in the rabbit and rat carotid bodies although at 2- and 4-fold lower levels respectively than that observed in the cat carotid body. Immunoelectron microscopy showed that NEP-specific immunoreactivity is associated with the intercellular region between the type I cells and cell clusters of the carotid body. Taken together, the results from this investigation demonstrate that neutral endopeptidase (EC 3.4.24.11) is one of the major endopeptidases which mediates the degradation and inactivation of neuropeptides in the carotid body.

Activation of nitric oxide synthase gene expression by hypoxia in central and peripheral neurons.

In the present study we examined the effects of hypobaric hypoxia on neuronal (n) and endothelial (e) nitric oxide synthase (NOS) gene expression in the central and peripheral nervous system. Adult rats were exposed either to normoxia (room air) on to hypobaric hypoxia (0.4 atm) for 4, 12 or 24 h and cerebellum and nodose ganglion representing the central and peripheral neurons, respectively, were removed. Messenger RNAs encoding n- and eNOS as well as beta-actin were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) technique. Hypoxia increased nNOS mRNA expression with maximal changes occurring after 12 h wherein mRNA levels were increased by 10.4 +/- 1.3 and 2 +/- 0.4 fold in nodose ganglion and cerebellum, respectively. Hypoxia, on the other hand, had no significant effect on eNOS and beta-actin mRNA levels. Analysis of nNOS protein and enzyme activity showed near doubling of these variables in both tissues after 24 h of hypoxia, indicating that nNOS protein levels are increased and that the protein is functionally active. These observations demonstrate that 12-24 h of hypobaric hypoxia selectively activates nNOS gene expression, which is reflected in an increase in nNOS protein in central and peripheral neurons. It is suggested that up-regulation of nNOS leads to increased generation of nitric oxide, which in turn may contribute to the readjustments of cardio-respiratory systems during the early stages of chronic hypoxia.

Nitric oxide synthase activity in guinea pig ventricular myocytes is not involved in muscarinic inhibition of cAMP-regulated ion channels.

It has recently been demonstrated that NO plays an obligatory role in muscarinic inhibition of beta-adrenergically stimulated ion channels in cardiac sinoatrial node cells (J Gen Physiol. 1995;106:45-65). We looked for evidence that NO might play a similar role in ventricular cells by using histochemical staining for NO synthase (NOS) activity and whole-cell patch-clamp recording of cAMP-regulated Cl- currents. Myocytes isolated from guinea pig hearts stained positively for NADPH-diaphorase activity, suggesting that these cells do express NOS. Acetylcholine (ACh) inhibition of the R(-)-isoproterenol bitartrate (Iso)-activated Cl- current was also reversed by the cGMP-lowering agents LY-83583 and methylene blue, consistent with idea that NO activation of guanylate cyclase may contribute to muscarinic responses. However, LY-83583 and methylene blue activated the Cl- current in the presence of subthreshold concentrations of Iso alone, suggesting that their effects may not be due to antagonism of an NO/cGMP-dependent response. Furthermore, ACh inhibition of Iso-activated Cl- currents could not be mimicked by the NO donors sodium nitroprusside,3-morpholinosydnonimine, and spermine-NO. Similarly, ACh inhibition of the Iso-activated Cl- current could not be blocked by the NOS inhibitor NG-monomethyl-L-arginine. These results indicate that even though ventricular myocytes possess NOS activity, NO production does not play an important role in muscarinic inhibition of beta-adrenergically regulated Cl- channels in these cells.

G proteins in carotid body chemoreception.

G proteins are signal coupling molecules that play major roles in mediating the effects of transmitters as well as certain sensory signals. In the present study we examined whether oxygen chemoreception in the carotid body is coupled to G proteins. Experiments were performed on carotid bodies isolated from anesthetized cats. Presence of G proteins was examined with ADP-ribosylation of the carotid body membranes. Pertussis toxin (PTX), which inactivates G proteins in neuronal tissues, ADP-ribosylated a single band of carotid body protein with a molecular mass of 41 kDa. With cholera toxin (CTX) only a faint band of protein corresponding to approximately 45 kDa was evident. Perfusing the isolated carotid bodies with PTX (2.5 micrograms/min; 60 min) attenuated the sensory response to hypoxia by 52% of the controls. Perfusion with CTX (50 micrograms/min; for 60 min), on the other hand, increased baseline activity and potentiated the hypoxic response by 125% of controls. Heat-inactivated toxins, however, had no influence on the carotid body sensory response to hypoxia. These results suggest that G proteins are present in the chemoreceptor tissue and they seem to be coupled to the transduction and/or to the transmission of the hypoxic stimulus.