Cough initiated from the trachea and larynx in anaesthetized guinea-pigs is mediated by capsaicin-insensitive, mechanically sensitive vagal afferent neurones. Tachykinin-containing, capsaicin-sensitive C-fibres also innervate the airways and have been implicated in the cough reflex. Capsaicin-sensitive nerves act centrally and synergistically to modify reflex bronchospasm initiated by airway mechanoreceptor stimulation. The hypothesis that polymodal mechanoreceptors and capsaicin-sensitive afferent nerves similarly interact centrally to regulate coughing was addressed in this study. Cough was evoked from the tracheal mucosa either electrically (16 Hz, 10 s trains, 1-10 V) or by citric acid (0.001-2 m). Neither capsaicin nor bradykinin evoked a cough when applied to the trachea of anaesthetized guinea-pigs, but they substantially reduced the electrical threshold for initiating the cough reflex. The TR<em>P</em>V1 receptor antagonist capsazepine prevented the increased cough sensitivity induced by capsaicin. These effects of topically applied capsaicin and bradykinin were not due to interactions between afferent nerve subtypes within the tracheal wall or a direct effect on the cough receptors, as they were mimicked by nebulizing 1 mg ml(-1) bradykinin into the lower airways and by microinjecting 0.5 nmol capsaicin into nucleus of the solitary tract (nTS). Citric acid-induced coughing was also potentiated by inhalation of bradykinin. The effects of tracheal capsaicin challenge on cough were mimicked by microinjecting <em>substance</em> <em>P</em> (0.5-5 nmol) into the nTS and prevented by intracerebroventricular administration (20 nmol h(-1)) of the neurokinin receptor antagonists C<em>P</em>99994 or SB223412. Tracheal application of these antagonists was without effect. C-fibre activation may thus sensitize the cough reflex via central mechanisms.

BACKGROUND

Depressed mood has been associated with decreased white matter and reduced hippocampal volumes. However, the relationship between brain structure and mood may be unique among adolescents who use marijuana heavily. The goal of this study was to examine the relationship between white matter and hippocampal volumes and depressive symptoms among adolescent marijuana users and controls.

METHODS

Data were collected from marijuana users (n = 16) and demographically similar controls (n = 16) aged 16-18. Extensive exclusionary criteria included psychiatric and neurologic disorders, including major depression. Substance use, mood, and anatomical measures were collected after 28 days of monitored abstinence.

RESULTS

Marijuana (MJ) users demonstrated more depressive symptoms than controls (p < .05). MJ use (beta = .42, p < .005) and smaller white matter volume (beta = -.34, p < .03) each predicted higher levels of depressive symptoms on the Hamilton Depression Rating Scale. MJ use interacted with white matter volume (beta = -.55, p < .03) in predicting depression scores on the Beck Depression Inventory: among MJ users, but not controls, white matter volume was negatively associated with depressive symptoms.

CONCLUSIONS

Marijuana use and white matter volume were additive and interactive in predicting depressive symptoms among adolescents. Subtle neurodevelopmental white matter abnormalities may disrupt the connections between areas involved in mood regulation.

Human organic anion transporter 4 (hOAT4) is located at the apical membrane of proximal tubule cells and involved in renal secretion and reabsorption of endogenous substances as well as many drugs and xenobiotics. This study reevaluated the physiologic role, transport mode, and driving forces of hOAT4. 6-Carboxyfluorescein (6-CF) uptake into HEK293 cells that stably expressed hOAT4 was saturable, resulting in a K(m) of 108 muM. 6-CF as well as [(3)H]estrone sulfate ([(3)H]ES) accumulation by HEK293-hOAT4 cells were abolished by ES, dehydroepiandrosterone sulfate, sulfinpyrazone, benzbromarone, and probenecid, whereas several OA, including p-aminohippurate (PAH), lactate, pyrazinoate, nicotinate, glutarate, and the diuretic hydrochlorothiazide (HCTZ) exhibited a slight or a NS inhibitory effect. PAH and glutarate are not taken up by HEK293-hOAT4 cells, but they trans-stimulated 6-CF and [(3)H]ES uptake, indicating an asymmetric interaction of hOAT4 with these substrates. In chloride-free medium, HEK293-hOAT4-mediated [(3)H]PAH efflux was almost abolished, whereas addition of ES restored it comparable to Ringer solution, consistent with a physiologic ES/PAH or PAH/Cl(-) exchange mode of hOAT4. Moreover, an acidification of the uptake medium increased 6-CF as well as [(3)H]ES uptake, which was reduced by nigericin, suggesting that hOAT4 also can operate as an OA/OH(-) exchanger. hOAT4 facilitates substantial uptake of [(14)C]urate, which was elevated 2.6-fold by intracellular HCTZ. Thus, hOAT4 is the long-postulated, low-affinity apical urate anion exchanger that facilitates HCTZ-associated hyperuricemia.

Abnormal membrane phospholipid essential polyunsaturated fatty acid (EPUFA) metabolism (i.e., reduced incorporation into phospholipids and increased breakdown) has been suggested to contribute to the etiopathophysiology of schizophrenia. However, most of the published studies have reported changes in the levels of membrane EPUFA in chronic medicated patients or in drug-naive patients long after onset of illness (1-2 years). Since the EPUFA metabolism can be altered by years of untreated illness or differentially altered by various antipsychotics, the significance of EPUFA membrane status to schizophrenia psychopathophysiology is unclear. We report the erythrocyte membrane EPUFA levels in drug-naive patients within +/- 4.5 days of onset of psychosis from an Army Medical Center, and in patients treated years with antipsychotics from a Veterans Affairs Medical Center. The levels of plasma lipid peroxides (TBARS, thiobarbituric acid reactive substances), products of damaged EPUFAs, were also determined. The levels of EPUFAs, particularly arachidonic acid (AA) and docosahexaenoic acid (DHA) were significantly lower (P < 0.001) in drug-naive patients at the onset of psychosis compared to matched normal controls. These lower EPUFA levels were associated with significantly higher levels of TBARS in patients (P < 0.001). The levels of AA and DHA were also lower (P < 0.001) and TBARS higher in chronic medicated patients than normal controls. However, the EPUFA levels were higher in chronic medicated patients than drug-naive first-episode patients. These data indicate that lower membrane AA and DHA most likely predate the illness and probably contribute to the onset of illness, and furthermore treatment with some antipsychotics may increase the levels of EPUFAs. The lipid peroxidation data suggest that possible increased oxidative stress, either as a part of the illness and/or its treatment with antipsychotics, may be one of the mechanisms of reduced membrane EPUFAs. These findings may have a significant impact on improving strategies for supplementation of EPUFAs and antioxidants to improve the outcome of schizophrenia.

BACKGROUND

It is unknown how genetic variants conferring liability to psychiatric disorders survive in the population despite strong negative selection. However, this is key to understanding their etiology and designing studies to identify risk variants.

OBJECTIVE

To examine the reproductive fitness of patients with schizophrenia and other psychiatric disorders vs their unaffected siblings and to evaluate the level of selection on causal genetic variants.

METHODS

We measured the fecundity of patients with schizophrenia, autism, bipolar disorder, depression, anorexia nervosa, or substance abuse and their unaffected siblings compared with the general population.

METHODS

Population databases in Sweden, including the Multi-Generation Register and the Swedish Hospital Discharge Register.

METHODS

In total, 2.3 million individuals among the 1950 to 1970 birth cohort in Sweden.

METHODS

Fertility ratio (FR), reflecting the mean number of children compared with that of the general population, accounting for age, sex, family size, and affected status.

RESULTS

Except for women with depression, affected patients had significantly fewer children (FR range for those with psychiatric disorder, 0.23-0.93; P < 10-10). This reduction was consistently greater among men than women, suggesting that male fitness was particularly sensitive. Although sisters of patients with schizophrenia and bipolar disorder had increased fecundity (FR range, 1.02-1.03; P < .01), this was too small on its own to counterbalance the reduced fitness of affected patients. Brothers of patients with schizophrenia and autism showed reduced fecundity (FR range, 0.94-0.97; P < .001). Siblings of patients with depression and substance abuse had significantly increased fecundity (FR range, 1.01-1.05; P < 10-10). In the case of depression, this more than compensated for the lower fecundity of affected individuals.

CONCLUSIONS

Our results suggest that strong selection exists against schizophrenia, autism, and anorexia nervosa and that these variants may be maintained by new mutations or an as-yet unknown mechanism. Bipolar disorder did not seem to be under strong negative selection. Vulnerability to depression, and perhaps substance abuse, may be preserved by balancing selection, suggesting the involvement of common genetic variants in ways that depend on other genes and on environment.

The mechanisms by which phosphorus homeostasis is preserved in mammals are not completely understood. We demonstrate the presence of a mechanism by which the intestine detects the presence of increased dietary phosphate and rapidly increases renal phosphate excretion. The mechanism is of physiological relevance because it maintains plasma phosphate concentrations in the normal range after ingestion of a phosphate-containing meal. When inorganic phosphate is infused into the duodenum, there is a rapid increase in the renal fractional excretion of phosphate (FE Pi). The phosphaturic effect of intestinal phosphate is specific for phosphate because administration of sodium chloride does not elicit a similar response. Phosphaturia after intestinal phosphate administration occurs in thyro-parathyroidectomized rats, demonstrating that parathyroid hormone is not essential for this effect. The increase in renal FE Pi in response to the intestinal administration of phosphate occurs without changes in plasma concentrations of phosphate (filtered load), parathyroid hormone, FGF-23, or secreted frizzled related protein-4. Denervation of the kidney does not attenuate phosphaturia elicited after intestinal phosphate administration. Phosphaturia is not elicited when phosphate is instilled in other parts of the gastrointestinal tract such as the stomach. Infusion of homogenates of the duodenal mucosa increases FE Pi, which demonstrates the presence of one or more substances within the intestinal mucosa that directly modulate renal phosphate reabsorption. Our experiments demonstrate the presence of a previously unrecognized phosphate gut-renal axis that rapidly modulates renal phosphate excretion after the intestinal administration of phosphate.

Peripheral and central injections of interleukin-1 (IL-1) and lipopolysaccharide (LPS) induce the expression of proinflammatory cytokines in the brain and have profound depressing effects on spontaneous and learned behaviors. These effects are mediated by vagal afferents, because they are abrogated by section of the vagus nerves at the subdiaphragmatic level in rats and mice. Vagotomy does not interfere with the synthesis and release of proinflammatory cytokines at the periphery, because plasma and tissue levels of interleukin-1 of vagotomized animals are similar to those of sham-operated animals. Furthermore, the consequences of vagotomy on the host behavioral response to peripheral cytokines are specific to the intraperitoneal route of administration of cytokines because vagotomized animals are still able to respond to IL-1 injected intravenously, subcutaneously, and into the lateral ventricle of the brain. Finally, substance P and cholecystokinin do not appear to play a key role in the transmission of the immune message to the brain because pretreatment by capsaicin or by specific antagonists of CCKA and CCKB receptors does not alter the behavioral effects of LPS and IL-1. All these findings point to the role of neural afferents for transmitting the immune message from the periphery to the brain.

In recent years, studies have advocated neuropeptide systems as modulators for the behavioral states found in mood disorders such as depression and anxiety disorders. Neuropeptides have been tested in traditional animal models and screening procedures that have been validated by known antidepressants and anxiolytics. However, it has become clear that although these tests are very useful, neuropeptides have distinct behavioral effects and dose-dependent characteristics, and therefore, use of these tests with neuropeptides must be done with an understanding of their unique characteristics. This review will focus on the behavioral actions of neuropeptides and their synthetic analogs, particularly in studies utilizing various preclinical tests of depression and anxiety. Specifically, the following neuropeptide systems will be reviewed: corticotropin-releasing factor (CRF), urocortin (Ucn), teneurin C-terminal associated peptide (TCAP), neuropeptide Y (NPY), arginine vasopressin (AVP), oxytocin, the Tyr-MIF-1 family, cholecystokinin (CCK), galanin, and substance P. These neuropeptide systems each have a unique role in the regulation of stress-like behavior, and therefore provide intriguing therapeutic targets for mood disorder treatment.

The vascular endothelium is an important mediator of tissue vasodilatation, yet the role of the specific substances, nitric oxide (NO) and prostaglandins (PG), in mediating the large increases in muscle perfusion during exercise in humans is unclear. Quadriceps microvascular blood flow was quantified by near infrared spectroscopy and indocyanine green in six healthy humans during dynamic knee extension exercise with and without combined pharmacological inhibition of NO synthase (NOS) and PG by L-NAME and indomethacin, respectively. Microdialysis was applied to determine interstitial release of PG. Compared to control, combined blockade resulted in a 5- to 10-fold lower muscle interstitial PG level. During control incremental knee extension exercise, mean blood flow in the quadriceps muscles rose from 10 +/- 0.8 ml (100 ml tissue)(-1) min(-1) at rest to 124 +/- 19, 245 +/- 24, 329 +/- 24 and 312 +/- 25 ml (100 ml tissue)(-1) min(-1) at 15, 30, 45 and 60 W, respectively. During inhibition of NOS and PG, blood flow was reduced to 8 +/- 0.5 ml (100 ml tissue)(-1) min(-1) at rest, and 100 +/- 13, 163 +/- 21, 217 +/- 23 and 256 +/- 28 ml (100 ml tissue)(-1) min(-1) at 15, 30, 45 and 60 W, respectively (P < 0.05 vs. control). In conclusion, combined inhibition of NOS and PG reduced muscle blood flow during dynamic exercise in humans. These findings demonstrate an important synergistic role of NO and PG for skeletal muscle vasodilatation and hyperaemia during muscular contraction.

OBJECTIVE

Bipolar disorder is associated with a high frequency of both completed suicides and suicide attempts. The primary aim of this study was to identify clinical predictors of suicide attempts in subjects with bipolar disorder.

METHODS

We studied 336 subjects with a diagnosis of bipolar I, bipolar II, or schizoaffective disorder (bipolar type). The Structured Clinical Interview for DSM-IV (SCID-I) was administered and subsequently two expert psychiatrists established a diagnosis. Predictors of suicide attempts were examined in attempters and non-attempters.

RESULTS

The lifetime rate of suicide attempts for the entire sample was 25.6%. A lifetime co-morbid substance use disorder was a significant predictor of suicide attempts: bipolar subjects with co-morbid substance use disorders (SUD) had a 39.5% lifetime rate of attempted suicide, while those without had a 23.8% rate (odds ratio = 2.09, 95% CI = 1.03-4.21, chi2 = 4.33, df = 1, p = 0.037).

CONCLUSIONS

Lifetime co-morbid SUD were associated with a higher rate of suicide attempts in patients with bipolar disorder. This relationship may have a genetic origin and/or be explained by severity of illness and trait impulsivity.

BACKGROUND

More than half of all individuals with bipolar disorder have a substance abuse problem at some point in their lifetime. Patients with comorbid substance abuse disorders often are excluded from clinical trials. Thus, treatments targeting this high-risk clinical population are lacking.

OBJECTIVE

To evaluate the efficacy of divalproex sodium (hereafter referred to as valproate) in decreasing alcohol use and stabilizing mood symptoms in acutely ill patients with bipolar disorder and alcoholism.

METHODS

A 24-week, double-blind, placebo-controlled, randomized parallel-group trial.

METHODS

A university hospital serving as a primary catchment-area hospital and tertiary-care facility.

METHODS

Fifty-nine subjects with diagnoses of bipolar I disorder and alcohol dependence. Intervention All study subjects received treatment as usual, including lithium carbonate and psychosocial interventions, and were randomized to receive valproate or placebo.

METHODS

Primary alcohol use outcomes included changes in alcohol use as indicated by changes in proportion of heavy drinking days and number of drinks per heavy drinking day. Other alcohol use outcomes included proportion of any drinking days, number of drinks per drinking day, and relapse to sustained heavy drinking. Mood outcomes included changes in depressive and manic symptoms. We used the mixed model to analyze longitudinal data. The first model used time of assessment, bipolar subtype (mixed, manic, or depressed), and treatment group (placebo or valproate) as covariates. The second nested model included the additional covariate of medication adherence.

RESULTS

The valproate group had a significantly lower proportion of heavy drinking days (P = .02) and a trend toward fewer drinks per heavy drinking day (P = .055) than the placebo group. When medication adherence was added as covariate, the valproate group had significantly fewer drinks per heavy drinking day (P = .02) and fewer drinks per drinking day (P = .02). Higher valproate serum concentration significantly correlated with improved alcohol use outcomes. Manic and depressive symptoms improved equally in both groups. Level of gamma-glutamyl transpeptidase was significantly higher in the placebo group compared with the valproate group.

CONCLUSIONS

Valproate therapy decreases heavy drinking in patients with comorbid bipolar disorder and alcohol dependence. The results of this study indicate the potential clinical utility of the anticonvulsant mood stabilizer, valproate, in bipolar disorder with co-occurring alcohol dependence.

Many different in vivo and in vitro tests are currently used to assess the toxicity of chemicals and complex mixtures such as cigarette smoke condensate. In vivo tests include assays in rodents to determine carcinogenicity, tumorigenicity and reproductive effects In vitro tests of mutagenicity are conducted with both bacterial and mammalian cell systems. A first step towards lowering the toxicity of cigarette smoke condensate is the identification of the relevant compound However, changing the concentration of a given smoke component may not linearly alter the biological activity of the complex mixture due to interactive effects. The "effective toxicity" of a chemical constituent is a function of the concentration, the metabolic fate, the potency in in vivo and in vitro assays, and the ability to reach the target tissues. The logarithm of the octanol-water partition coefficient (log P) is an important parameter since it affects metabolism, biological transport properties and intrinsic toxicity. Using concentration data from the International Agency for Cancer Research (IARC), biological activity data from the Registry of Toxic Effects of Chemical Substances (RTECS) database and measured and calculated log P values, we have rank ordered some of the important compounds in cigarette smoke condensate by their measured or potential toxicity. Condensates from different cigarette brands, tar categories and styles vary in their concentrations of these compounds. Chemicals of greater commercial or scientific interest may be toxicity tested more extensively, thereby increasing the probability of positive test results and highlighting the need for consideration of structure-activity relationships.

The estimation of the accuracy of predictions is a critical problem in QSAR modeling. The "distance to model" can be defined as a metric that defines the similarity between the training set molecules and the test set compound for the given property in the context of a specific model. It could be expressed in many different ways, e.g., using Tanimoto coefficient, leverage, correlation in space of models, etc. In this paper we have used mixtures of Gaussian distributions as well as statistical tests to evaluate six types of distances to models with respect to their ability to discriminate compounds with small and large prediction errors. The analysis was performed for twelve QSAR models of aqueous toxicity against T. pyriformis obtained with different machine-learning methods and various types of descriptors. The distances to model based on standard deviation of predicted toxicity calculated from the ensemble of models afforded the best results. This distance also successfully discriminated molecules with low and large prediction errors for a mechanism-based model developed using log P and the Maximum Acceptor Superdelocalizability descriptors. Thus, the distance to model metric could also be used to augment mechanistic QSAR models by estimating their prediction errors. Moreover, the accuracy of prediction is mainly determined by the training set data distribution in the chemistry and activity spaces but not by QSAR approaches used to develop the models. We have shown that incorrect validation of a model may result in the wrong estimation of its performance and suggested how this problem could be circumvented. The toxicity of 3182 and 48774 molecules from the EPA High Production Volume (HPV) Challenge Program and EINECS (European chemical Substances Information System), respectively, was predicted, and the accuracy of prediction was estimated. The developed models are available online at http://www.qspr.org site.

Information regarding the prevalence of mental illness and substance use among HIV-infected patients and the effect of these problems on HIV treatment is needed. We conducted an observational study of patients in the University of Washington (UW) HIV Cohort to determine prevalence rates for mental illness and substance use. Cox regression analyses were used to examine the relationship between mental illness and substance use, pharmacologic treatment for depression/anxiety, and initiation of highly active antiretroviral therapy (HAART) within 9 months of becoming eligible for HAART. Among 1774 patients in the UW HIV cohort during 2004, 63% had a mental illness (including mood, anxiety, psychotic, or personality disorders), 45% had a substance use disorder, and 38% had both. There were 278 patients who met criteria for HAART eligibility. After controlling for other factors, patients with depression and/or anxiety were significantly less likely to initiate HAART compared with patients without a mental illness (hazard ratio [HR] 0.4, p = 0.02). However, patients with depression/anxiety who received antidepressant/antianxiety medications were equally likely to initiate HAART as patients without a mental illness (HR 0.9, p = 0.5). We found that patients with mental illness or substance use disorders receive HAART at lower CD4+ cell counts and higher HIV-1 RNA levels than patients without these disorders. However, HAART initiation among patients who receive treatment for depression/anxiety is associated with no delay. Screening for these disorders in primary care settings and access to appropriate treatment are increasingly important components of providing care to HIV-infected patients.

OBJECTIVE

This study compared sexual risk behaviors of gay, lesbian, and bisexual (GLB) and heterosexual adolescents and evaluated associations between gay-sensitive HIV instruction and risk behaviors of GLB youths.

METHODS

A random sample of high school students and HIV education teachers completed surveys. Self-reported risk behaviors of heterosexual and GLB adolescents were compared, with control for student and community demographic characteristics. Sexual risk behaviors of GLB youths in schools with and without gay-sensitive instruction were compared.

RESULTS

GLB youths reported more substance use, high-risk sexual behaviors, suicidal thoughts or attempts, and personal safety issues than did heterosexual youths (P < .001). Among those who were sexually active, GLB youths reported more lifetime and recent sexual partners than did heterosexuals (P < .001), and more of them reported alcohol use before last sex (P < .01) and a history of pregnancy (P < .001). GLB youths in schools with gay-sensitive instruction reported fewer sexual partners, less recent sex, and less substance use before last sex than did GLB youths in other schools (P < .05).

CONCLUSIONS

The findings document increased risk behaviors among GLB youths and demonstrate the potential benefits of providing gay-sensitive HIV instruction in schools.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Multiple Sclerosis (MS) are characterized by neuronal degeneration and neuronal death in specific regions of the central nervous system (CNS). In AD, neurons of the hippocampus and entorhinal cortex are the first to degenerate, whereas in PD, dopaminergic neurons in the substantia nigra degenerate. MS patients show destruction of the myelin sheath. Once the CNS neurons are damaged, they are unable to regenerate unlike any other tissue in the body. Neurodegeneration is mediated by inflammatory and neurotoxic mediators such as interleukin-1beta (IL-1β), IL-6, IL-8, IL-33, tumor necrosis factor-alpha (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), CCL5, matrix metalloproteinase (MMPs), granulocyte macrophage colony-stimulating factor (GM-CSF), glia maturation factor (GMF), substance P, reactive oxygen species (ROS), reactive nitrogen species (RNS), mast cells-mediated histamine and proteases, protease activated receptor-2 (PAR-2), CD40, CD40L, CD88, intracellular Ca+ elevation, and activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-B (NF-kB). Activated microglia, astrocytes, neurons, T-cells and mast cells release these inflammatory mediators and mediate neuroinflammation and neurodegeneration in a vicious manner. Further, immune and inflammatory cells and inflammatory mediators from the periphery cross the defective blood-brain-barrier (BBB) and augment neuroinflammation. Though inflammation is crucial in the onset and the progression of neurodegenerative diseases, anti-inflammatory drugs do not provide significant therapeutic effects in these patients till date, as the disease pathogenesis is not yet clearly understood. In this review, we discuss the possible factors involved in neuroinflammation-mediated neurodegeneration.

OBJECTIVE

This study evaluated the effectiveness of targeted televised public service announcement campaigns in reducing marijuana use among high-sensation-seeking adolescents.

METHODS

The study used a controlled interrupted time-series design in 2 matched communities. Two televised antimarijuana campaigns were conducted in 1 county and 1 campaign in the comparison community. Personal interviews were conducted with 100 randomly selected teenagers monthly in each county for 32 months.

RESULTS

All 3 campaigns reversed upward developmental trends in 30-day marijuana use among high-sensation seekers (P < .002). As expected, low-sensation seekers had low use levels, and no campaign effects were evident.

CONCLUSIONS

Televised campaigns with high reach and frequency that use public service announcements designed for and targeted at high-sensation-seeking adolescents can significantly reduce substance use in this high-risk population.

We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Some vagal afferent nerves are thought to mediate autonomic responses evoked by noxious oesophageal stimuli and participate in the perception of pain originating in the oesophagus. However, the vagal nociceptive nerve phenotypes implicated in this function have yet to be identified. In this study, nociceptive fibres were defined by the capacity to discriminate noxious mechanical stimuli (wide range of oesophageal distension with pressure up to 100 mmHg) and detect noxious chemical stimuli (the activators of capsaicin receptor TRPV1). Using immunohistochemical techniques with retrogradely labelled oesophagus-specific neurones and performing extracellular recordings from the isolated vagally innervated oesophagus, we show that in the guinea-pig, the vagus nerves supply the oesophagus with a large population of nociceptive-like afferent nerve fibres. Vagal nociceptive-like fibres in the guinea-pig oesophagus are derived from two embryonically distinct sources: neurones situated in the nodose vagal ganglia and neurones situated in the jugular vagal ganglia. Nodose (placode-derived) nociceptive-like fibres are exclusively C-fibres sensitive to a Psubstance P. In contrast, jugular (neural crest-derived) nociceptive-like fibres include both A-fibres and C-fibres, are insensitive to Psubstance P. The non-nociceptive vagal tension mechanoreceptors are distinguished from nociceptors by their saturable response to oesophageal distension and by the lack of TRPV1. These tension mechanoreceptors are exclusively A-fibres arising from the nodose ganglion. We conclude that the vagus nerves supply the guinea-pig oesophagus with nociceptors in addition to tension mechanoreceptors. The vagal nociceptive-like fibres in the oesophagus comprise two distinct subtypes dictated by the ganglionic location of their cell bodies.

BACKGROUND

Although pharmacological studies suggest that the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in sensory nerve fibers innervating the heart may exert a cardioprotective effect, definitive evidence supporting such a notion is lacking. In addition, function and regulation of sensory neuropeptides, namely, calcitonin gene-related peptide (CGRP) and substance P (SP), in the face of challenges induced by cardiac injury in the presence or absence of the TRPV1 are largely unknown.

RESULTS

The hearts of gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice or wild-type (WT) mice were perfused in a Langendorff apparatus in the presence or absence of capsazepine (a TRPV1 receptor antagonist), CGRP, CGRP(8-37) (a CGRP receptor antagonist), SP, or RPP release measured by radioimmunoassay were assessed before and after ischemia/reperfusion injury of the heart. Expression of the NK1 receptor in the hearts of TRPV1(-/-) and WT mice were determined with the use of Western blot analyses. Impairment of postischemic recovery, defined by increased left ventricular end-diastolic pressure (LVEDP) and decreased left ventricular developed pressure (LVDP) and coronary flow (CF), was more severe in TRPV1(-/-) hearts than in WT hearts. Although it had no effect on postischemic recovery of TRPV1(-/-) hearts, blockade of the TRPV1 with capsazepine caused a most severe impairment of postischemic recovery in WT hearts compared with untreated WT and TRPV1(-/-) hearts. Exogenous CGRP and SP produced a significant improvement in postischemic recovery in both TRPV1(-/-) and WT hearts, and the maximal functional improvement in TRPV1(-/-) hearts was not different from that of WT hearts except that SP-induced increases in LVDP were larger in the former than in the latter. Blockade of the NK1 receptor with RPP receptor with CGRP(8-37), caused more severe impairment in postischemic recovery in both TRPV1(-/-) and WT hearts than in untreated hearts in both genotypes. The release of SP after ischemia/reperfusion injury was increased in both WT and TRPV1(-/-) hearts, albeit with a smaller magnitude of the increase in the latter. Capsazepine attenuated injury-induced SP release in WT but not TRPV1(-/-) hearts. There was no difference in the expression of the NK1 receptor between the 2 genotype hearts.

CONCLUSIONS

Thus, our data show that (1) TRPV1 gene deletion decreases injury-induced SP release and impairs cardiac recovery function after ischemia/reperfusion injury; (2) TRPV1 gene deletion leads to reconditioning of the heart with improved postischemic recovery compared with that induced by acute TRPV1 blockade and in terms of cardiac response to exogenous SP; and (3) blockade of the NK1 but not CGRP receptors worsens postischemic recovery of hearts in both genotypes. Taken together, these data indicate that TRPV1 plays a role in protecting the heart from injury possibly via increasing SP release and that deletion of this receptor reconditions the heart for escaping, at least in part, from injury possibly via enhancing NK1 receptor function.

Arylsulfatase preferentially present in the human eosinophil as compared to other leukocytes was isolated by sequential gel filtration and cation exchange chromatography. The apparent molecular weight of 60,000, the preferential cleavage of 4-nitrocatechol sulfate (PNCS) over p-acetyl-benzenesulfonic acid (PABS), inhibition by phosphate ions and pH optimum of 5.7 are characteristics of a type II B arylsulfatase. Eosinophil arylsulfatase inactivated purified human slow reacting substance of anaphylaxis (SRS-A) in a time-dependent reaction with the rate dependent upon the enzyme/substrate ratio. That SRS-A inactivation was the result of intrinsic arylsulfatase activity was indicated by association of PNCS cleavage and SRS-A inactivating activity during chromatography, the similar pH optimum for cleavage of both substrates and the capacity of SRS-A to inhibit PNCS cleavage by arylsulfatase. The finding that eosinophil arylsulfatase inactivates SRS-A suggests that eosinophil ingress into the site of an immediate hypersensitivity reaction in response to ECF-A could represent a regulatory function.

We have investigated the possible effect of substance P (SP), a main mediator of neurogenic inflammation, on the growth of capillary vessels in vivo, and on the proliferation of cultured endothelial cells in vitro. Slow release preparations of SP were implanted into the avascular cornea of New Zealand White rabbits and vessel growth was monitored daily through a slit lamp stereomicroscope. SP (1-5 micrograms/pellet) induced a marked neovascularization. A selective NK-1 receptor agonist [beta-Ala4, Sar9, Met(O2)11]-SP(4-11) also induced neovascularization. The addition of SP to serum-free cultured endothelial cells, isolated from bovine adrenals (BACE) and from human umbilical cord veins (HUVE), increased proliferation of both cell lines in a concentration-dependent manner with maximal activity at 10(-8) M (BACE) and 10(-10) M (HUVE). The selective NK-1 receptor agonist induced a similar proliferative action on both cell lines, while the selective NK-2 receptor agonist [beta-Ala8]-NKA(4-10) and the selective NK-3 receptor agonist [MePhe7]-NKB had no significant effect. Two different SP antagonists [D-Pro2, D-Trp7,9]-SP and [D-Pro4, D-Trp7,9,Phe11]-SP (4-11) blocked the response to SP. These findings indicate that SP can directly stimulate the process of neovascularization, probably through induction of endothelial cell proliferation. This hitherto unraveled activity of SP could play a key role in the trophic action produced by activation of the efferent function of peripheral endings of primary sensory neurons.

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.

Neurotransmitters have been shown to coexist in cell bodies, but demonstrating their coexistence within nerve fibers and terminals has been more difficult. However, two recent reports outlined a simple light-microscopic method by which two neurotransmitters can be shown to coexist in fibers and terminals. The method was identical to that used for immunohistochemical localization of one antigen, except that two primary--secondary antibody systems labeled with two different fluorochromes were used simultaneously. In the present study, a method for the simultaneous visualization of serotonin and substance P was characterized. This method employed an antiserum to serotonin generated in goat in combination with a rabbit-generated antiserum to substance P. These antisera were visualized with secondary antisera raised in swine and conjugated with rhodamine and fluorescein respectively. Spinal cord sections stained by this protocol showed large numbers of fibers fluorescing both red and green. Many of them were in the ventral horn, fewer were around the central canal, and virtually none were in the dorsal horn. The apparent double labeling could be shown not to be the result of cross-reactivity among the antisera, of any inappropriate affinity among the antisera, of green fluorescence by rhodamine, or of red fluorescence by fluorescein. It is concluded that the method provides a simple technique for visualizing fibers and terminals in which serotonin and substance P coexist.