Involvement of capsaicin-sensitive sensory neurons in gastrointestinal function

There is both morphological and functional evidence that capsaicin-sensitive sensory neurons innervate the digestive tract. The possible function of these neurons in gastric ulceration and gastrointestinal motility was investigated in rats which had been systemically pretreated with capsaicin (50-125 mg/kg). It was found that capsaicin-sensitive afferent neurons do not participate in the physiologic control of gastrointestinal propulsion. However, the inhibition of gastrointestinal transit due to surgical trauma or peritoneal irritation with iodine was reduced in capsaicin-treated rats. It was concluded that capsaicin-sensitive sensory neurons may be involved in sympathetic reflex inhibition of gastrointestinal propulsion. Gastric ulceration induced by the intraperitoneal injection of indomethacin or intragastric administration of ethanol was greatly aggravated in capsaicin-treated rats. Since an involvement of the autonomic nervous system as well as of histamine and prostaglandins in this effect of capsaicin treatment could be ruled out, further support was lent to the previously proposed hypothesis that sensory nerve endings can protect the gastric mucosa against ulceration by the local release of vasodilator substances.     

Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1alpha, and PGE2 were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with L-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2 and PGE2 by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.     

Involvement of capsaicin-sensitive sensory nerves in cardioprotection of rutaecarpine in rats

In the present study, we examined whether rutaecarpine protects against myocardial ischemia-reperfusion injury in rats and whether the protective effects of rutaecarpine are related to activation of capsaicin-sensitive sensory nerves. Rats were pretreated with rutaecarpine 10 min before the experiment, and then the left main coronary artery of rat hearts was subjected to 60-min occlusion followed by 3-h reperfusion. The infarct size, serum concentration of creatine kinase, and CGRP concentration in plasma were measured. Pretreatment with rutaecarpine (100 or 300 microg/kg, i.v.) significantly reduced infarct size and creatine kinase release concomitantly with a significant increase in plasma concentrations of CGRP. These effects of rutaecarpine were completely abolished by capsazepine (38 mg/kg, s.c.), a competitive vanilloid receptor antagonist, or by pretreatment with capsaicin (50 mg/kg, s.c.), which selectively depletes transmitters in capsaicin-sensitive sensory nerves. These results suggest that rutaecarpine protects against myocardial ischemia-reperfusion injury in rats and that the protective effects of rutaecarpine are related to activation of capsaicin-sensitive sensory nerves via activating vanilloid receptors.     

Impaired glucose homeostasis in mice lacking the alpha1b-adrenergic receptor subtype

To assess the role of the alpha1b-adrenergic receptor (AR) in glucose homeostasis, we investigated glucose metabolism in knockout mice deficient of this receptor subtype (alpha1b-AR-/-). Mutant mice had normal blood glucose and insulin levels, but elevated leptin concentrations in the fed state. During the transition to fasting, glucose and insulin blood concentrations remained markedly elevated for at least 6 h and returned to control levels after 24 h whereas leptin levels remained high at all times. Hyperinsulinemia in the post-absorptive phase was normalized by atropine or methylatropine indicating an elevated parasympathetic activity on the pancreatic beta cells, which was associated with increased levels of hypothalamic NPY mRNA. Euglycemic clamps at both low and high insulin infusion rates revealed whole body insulin resistance with reduced muscle glycogen synthesis and impaired suppression of endogenous glucose production at the low insulin infusion rate. The liver glycogen stores were 2-fold higher in the fed state in the alpha1b-AR-/- compared with control mice, but were mobilized at the same rate during the fed to fast transition or following glucagon injections. Finally, high fat feeding for one month increased glucose intolerance and body weight in the alpha1b-AR-/-, but not in control mice. Altogether, our results indicate that in the absence of the alpha1b-AR the expression of hypotalamic NPY and the parasympathetic nervous activity are both increased resulting in hyperinsulinemia and insulin resistance as well as favoring obesity and glucose intolerance development during high fat feeding.     

Hypoglycemic detection at the portal vein is mediated by capsaicin-sensitive primary sensory neurons

To elucidate the type of spinal afferent involved in hypoglycemic detection at the portal vein, we considered the potential role of capsaicin-sensitive primary sensory neurons. Specifically, we examined the effect of capsaicin-induced ablation of portal vein afferents on the sympathoadrenal response to hypoglycemia. Under anesthesia, the portal vein was isolated in rats and either capsaicin (CAP) or the vehicle (CON) solution applied topically. During the same surgery, the carotid artery (sampling) and jugular vein (infusion) were cannulated. One week later, all animals underwent a hyperinsulinemic hypoglycemic clamp, with glucose (variable) and insulin (25 mU x kg(-1) x min(-1)) infused via the jugular vein. Systemic hypoglycemia (2.76 +/- 0.05 mM) was induced by minute 75 and sustained until minute 105. By design, no significant differences were observed in arterial glucose or insulin concentrations between groups. When hypoglycemia was induced in CON, the plasma epinephrine concentration increased from 0.67 +/- 0.05 nM at basal to 36.15 +/- 2.32 nM by minute 105. Compared with CON, CAP animals demonstrated an 80% suppression in epinephrine levels by minute 105, 7.11 +/- 0.55 nM (P < 0.001). A similar response to hypoglycemia was observed for norepinephrine, with CAP values suppressed by 48% compared with CON. Immunohistochemical analysis of the portal vein revealed an 85% decrease in the number of calcitonin gene-related peptide-reactive nerve fibers following capsaicin-induced ablation. That the suppression in the sympathoadrenal response was comparable to our previous findings for total denervation of the portal vein indicates that hypoglycemic detection at the portal vein is mediated by capsaicin-sensitive primary sensory neurons.     

Functional interaction between transforming growth factor alpha and capsaicin-sensitive sensory neurons in the rat stomach

BACKGROUND AND AIMS: Transforming growth alpha (TGFalpha) and sensory neurons have been shown to promote gastric mucosal protection and healing. Aims were to examine in vitro interactions between gastric sensory neurons, the sensory neuropeptide calcitonin gene-related peptide (CGRP), and TGFalpha. METHODS: Gastric mucosal/submucosal tissue fragments from Sprague-Dawley (SD) rats were incubated in short-term (30 min) culture. Peptide release into media and TGFalpha tissue content were measured by radioimmunoassay. RESULTS: TGFalpha (1 x 10(-8) to 1 x 10(-6) M) caused dose-dependent stimulation of CGRP release. Maximal CGRP release (+87%) was observed with 1 x 10(-6) M TGFalpha: 28.6+/-3.8 vs. control of 15.5+/-2.7 pg/g tissue; P<0.05. Both CGRP (1 x 10(-7) to 1 x 10(-5) M) and capsaicin (1 x 10-(8) to 1 x 10(-6)M) significantly inhibited basal TGFalpha release in a dose-dependent fashion that ranged from -20% to -39%. In contrast, capsaicin-induced sensory denervation caused significant increases in both basal TGFalpha release and TGFalpha tissue content. CONCLUSION: Function interactions between TGFalpha and gastric sensory neurons are suggested by the observations that (1) TGFalpha stimulated CGRP release from gastric sensory neurons; (2) CGRP and acute capsaicin treatment inhibited TGFalpha release and; (3) capsaicin-induced sensory denervation caused significant increases in both gastric TGFalpha basal release and tissue content.     

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.     

Coexisting peptides in capsaicin-sensitive sensory neurons: release and actions in the respiratory tract of the guinea-pig

Release of substance P (SP) and neurokinin A (NKA), was demonstrated in the isolated perfused guinea-pig lung. Significant release was obtained by perfusion with capsaicin, high potassium, histamine, bradykinin dimethylphenylpiperazinium, and by electrical vagal nerve stimulation. Capsaicin-induced peptide release was not blocked by 1 microM clonidine. SP and NKA contracted respiratory smooth muscle, NKA being 42 times more potent. Both tachykinins were equipotent in relaxing pulmonary artery. It is concluded that multiple tachykinin can be released from capsaicin-sensitive sensory nerves in the respiratory tract, exerting multiple effects on the target tissues.     

Neurofilaments and microtubules in sensory neurons after peripheral nerve section

Summary Sensory neurons were examined in spinal ganglia of the rat 1 to 55 days after section of the plexus brachialis nerves. Only light neurons of the type A were investigated. Maximal reaction to axotomy was found 7 to 14 days after the operation. The majority of the axotomized perikarya developed central chromatolysis. In such neurons, Nissl bodies virtually disappeared from the central area of the neuron and formed a more or less continuous zone at the cell circumference. The cytocentrum became filled with large numbers of mitochondria, dense bodies and other organelles. Neurofilaments and microtubules were disarranged and ran at random among the accumulated particles. Microtubules were often more prominent in chromatolytic areas than neurofilaments. Both these organelles were rare in the peripheral areas filled with massed Nissl substance.Part of the neurons that did not show typical chromatolysis contained increased numbers of neurofilaments among Nissl bodies dispersed throughout the cytoplasm. Neurofilaments were roughly arrayed in bundles up to several microns wide; they were linked by cross-bridges and separated by distances of about 500 Å. Microtubules were rarely found in the filamentous areas. However, they were numerous in the axon hillock and in the initial segment where they formed fascicles similar to those described in normal neurons of other types.During the period from 14 to 55 days after axotomy, many perikarya recovering from chromatolysis contained enlarged bundles of neurofilaments with occasional microtubules among the restored Nissl bodies.Mean diameters of sensory neurons, measured 7 to 55 days after axotomy, in no instance exceeded those of contralateral control neurons. It thus appears that sensory perikarya do not increase in size either during the chromatolytic process or during the period of recovery.This project was supported by a grant from the Muscular Dystrophy Association of America, Inc. The main part of this study was done while the author was a Research Fellow in Anatomy at the Harvard Medical School, Boston. The author wishes to thank prof. S. L. Palay for his valuable advice and help received during her stay at the Department of Anatomy at the Harvard Medical School, under NIH training grant NBO5591.     

Transforming growth factor alpha-mediated gastroprotection against stress ulceration in the rat: involvement of capsaicin-sensitive sensory neurons

Exogenously administered TGF alpha has been shown to protect rodent gastric mucosa against injury caused by acid-dependent and acid-independent injury. The present study examined whether the gastroprotective effects of TGF alpha on stress-induced gastric ulceration in the rat involves activation of capsaicin-sensitive sensory neurons. Fasted male SD rats were subjected to water restraint stress (WRS) for four hours. Thereafter, rats were euthanized; the stomach opened and macroscopic areas of gastric ulceration quantitated (mm(2)). Gastric tissue contents of TGF alpha and the sensory neuropeptide, calcitonin gene-related peptide (CGRP) were determined by radioimmunoassay. Prior to stress rats received TGF alpha 50, 100 or 200 microg/kg by intraperitoneal injection. Sensory denervation was accomplished by high dose capsaicin treatment. WRS caused severe ulceration in the gastric corpus; 46.1 + 6.6 mm(2). Parenteral administration of TGF alpha caused dose-dependent reduction in gastric injury: 34.7 + 4.9 mm(2) with 50 microg/kg (p < 0.05); 25.4 + 3.6 mm(2) with 100 microg/kg (p < 0.001) and 9.4 + 0.8 mm(2) with 200 microg/kg (p < 0.001). The gastroprotective action of TGF alpha (200 microg/kg, i.p.) was abolished by capsaicin-induced sensory denervation. In addition, WRS ulceration was associated with significant reduction in gastric CGRP (-42%) and TGF alpha (-48%) content. Reduction in CGRP content was prevented by TGF alpha pretreatment. We conclude that: 1) TGF alpha caused dose-dependent gastroprotection against WRS ulceration, 2) TGF alpha-mediated gastric mucosal protection was prevented by capsaicin-induced sensory denervation and, 3) stress-induced injury was associated with significant reduction in gastric content of both TGF alpha and CGRP.     

Early peripheral sensory neurons in the development of trochozoan animals

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.     

Compensatory gastroprotective action of glucocorticoid hormones in the rats with ablation of capsaicin-sensitive neurons

We tested the hypothesis that contribution of glucocorticoids in gastroprotection become especially important during ablation of capsaicin-sensitive neurons. For this, the effect of desensitization of capsaicin-sensitive neurons on the gastric mucosa was compared in groups of rats with different glucocorticoid supply: sham-operated and adrenalectomized without and with corticosterone replacement (4 mg/kg sc). Functional ablation of capsaicin-sensitive neurons was performed with neurotoxic doses of capsaicin (20 + 30 + 50 mg/kg sc). Indomethacin in the dose 35 mg/kg was given as an ulcerogenic stimulus. It was shown that combination of adrenalectomy with desensitization of capsaicin-sensitive neurons potentiated the effect of sensory desensitization alone on indomethacin-induced gastric erosions. Corticosterone replacement prevented this effect of adrenalectomy. The results suggest a pivotal compensatory role of glucocorticoids in maintenance of gastric mucosal integrity during ablation of caspsaicin-sensitive sensory neurons.     

Impaired regenerative response of primary sensory neurons in ZPK/DLK gene-trap mice

Rapid and persistent activation of c-JUN is necessary for axonal regeneration after nerve injury, although upstream molecular events leading to c-JUN activation remain largely unknown. ZPK/DLK/MAP3K12 activates the c-Jun N-terminal kinase pathway at an apical level. We investigated axonal regeneration of the dorsal root ganglion (DRG) neurons of homozygous ZPK/DLK gene-trap mice. In vitro neurite extension assays using DRG explants from 14 day-old mice revealed that neurite growth rates of the ZPK/DLK gene-trap DRG explants were reduced compared to those of the wild-type DRG explants. Three ZPK/DLK gene-trap mice which survived into adulthood were subjected to sciatic nerve axotomy. At 24 h after axotomy, phosphorylated c-JUN-positive DRG neurons were significantly less frequent in ZPK/DLK gene-trap mice than in wild-type mice. These results indicate that ZPK/DLK is involved in regenerative responses of mammalian DRG neurons to axonal injury through activation of c-JUN.     

Impaired glucose homeostasis after a transient intermittent hypoxic exposure in neonatal rats

This initial report presents a neonatal rat model with exposure to a transient intermittent hypoxia (IH), which results in a persisting diabetes-like condition in the young rats. Twenty-five male pups were treated at postnatal day 1 with IH exposure by alternating the level of oxygen between 10.3% and 20.8% for 5 h. The treated animals were then maintained in normal ambient oxygen condition for 3 week and compared to age-matched controls. The IH treated animals exhibited a significantly higher fasting glucose level than the control animals (237.00 ± 19.66 mg/dL vs. 167.25 ± 2.95 mg/dL; P = 0.003); and a significantly lower insulin level than the control (807.0 ± 72.5 pg/mL vs. 1839.8 ± 377.6 pg/mL; P = 0.023). There was no difference in the mass or the number of insulin producing beta cells as well as no indicative of inflammatory changes; however, glucose tolerance tests showed a significantly disturbed glucose homeostasis. In addition, the amount of C-peptide secreted from the islets harvested from the IH animals were decreased significantly (from 914 pM in control to 809 pM in IH; P = 0.0006) as well. These observations demonstrate that the neonatal exposure to the IH regimen initiates the development of deregulation in glucose homeostasis without infiltration of inflammatory cells.     

Body temperature dependency in baclofen-induced gastric acid secretion in rats relation to capsaicin-sensitive afferent neurons

Body temperature dependency in gastric functional responses to baclofen, a GABA(B) agonist, such as acid secretion, mucosal blood flow (GMBF) and motor activity, was examined in urethane-anesthetized rats under normal (37+/-1 degrees C) and hypothermic (31+/-1 degrees C) conditions. A rat stomach was mounted in an ex-vivo chamber, perfused with saline, and the acid secretion was measured using a pH-stat method, simultaneously with GMBF by a laser Doppler flowmeter. Gastric motility was measured using a miniature balloon as intraluminal pressure recordings. Intravenous administration of baclofen significantly increased acid secretion at the doses > 0.3 mg/kg under hypothermic conditions, yet it caused a significant stimulation only at doses > 10 mg/kg under normothermic conditions. The increases in gastric motility and GMBF were similarly induced by baclofen, irrespective of whether the animals were subjected to normothermic or hypothermic conditions. These functional responses to baclofen under hypothermic conditions were totally attenuated by either bilateral vagotomy or atropine (3 mg/kg, s.c.). Baclofen at a lower dose (1 mg/kg i.v.) significantly increased the acid secretion even under normothermic conditions when the animals were subjected to chemical deafferenation of capsaicin-sensitive neurons or pretreatment with intracisternal injection of CGRP8-37 (30 ng/rat). These results suggest that 1) gastric effects of baclofen are dependent on body temperature in stimulating acid secretion but not GMBF or motor activity, 2) the acid stimulatory action of baclofen is enhanced under hypothermic conditions, and 3) the suppression of baclofen-induced acid response under normothermic conditions may be related to capsaicin-sensitive afferent neuronal activity, probably mediated by central release     

Reduced pain behaviors and extracellular signal-related protein kinase activation in primary sensory neurons by peripheral tissue injury in mice lacking platelet-activating factor receptor

Peripheral tissue injury causes the release of various mediators from damaged and inflammatory cells, which in turn activates and sensitizes primary sensory neurons and thereby produces persistent pain. The present study investigated the role of platelet-activating factor (PAF), a phospholipid mediator, in pain signaling using mice lacking PAF receptor (pafr-/- mice). Here we show that pafr-/- mice displayed almost normal responses to thermal and mechanical stimuli but exhibit attenuated persistent pain behaviors resulting from tissue injury by locally injecting formalin at the periphery as well as capsaicin pain and visceral inflammatory pain without any alteration in cytoarchitectural or neurochemical properties in dorsal root ganglion (DRG) neurons and a defect in motor function. However, pafr-/- mice showed no alterations in spinal pain behaviors caused by intrathecally administering agonists for N-methyl-d-aspartate (NMDA) and neurokinin(1) receptors. A PAFR agonist evoked an intracellular Ca(2+) response predominantly in capsaicin-sensitive DRG neurons, an effect was not observed in pafr-/- mice. By contrast, the PAFR agonist did not affect C- or Adelta-evoked excitatory post-synaptic currents in substantia gelatinosa neurons in the dorsal horn. Interestingly, mice lacking PAFR showed reduced phosphorylation of extracellular signal-related protein kinase (ERK), an important kinase for the sensitization of primary sensory neurons, in their DRG neurons after formalin injection. Furthermore, U0126, a specific inhibitor of the ERK pathway suppressed the persistent pain by formalin. Thus, PAFR may play an important role in both persistent pain and the sensitization of primary sensory neurons after tissue injury.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

Impaired host defense in mice lacking ONZIN

ONZIN is a small, cysteine-rich peptide of unique structure that is conserved in all vertebrates examined to date. We show that ONZIN is expressed at high levels in epithelial cells of the intestinal tract, the lung, and in cells of the immune system including macrophages and granulocytes. Because this pattern of expression is suggestive of a role in innate immune function, we have generated mice lacking this protein and examined their ability to respond to challenge with infectious agents. Onzin(-/-) mice show a heightened innate immune response after induction of acute peritonitis with Klebsiella pneumoniae. This increased response is consistent with an increased bacterial burden in the Onzin(-/-) mice. Ex vivo studies show that, whereas phagocytosis is not altered in Onzin(-/-) neutrophils, phagocytes lacking this protein kill bacteria less effectively. This result identifies ONZIN as a novel class of intracellular protein required for optimal function of the neutrophils after uptake of bacteria.     

Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.