Wolfgang Kummer

Induction of tachykinin gene and peptide expression in guinea pig nodose primary afferent neurons by allergic airway inflammation.

Substance P (SP), neurokinin A (NKA), and calcitonin gene-related peptide (CGRP) have potent proinflammatory effects in the airways. They are released from sensory nerve endings originating in jugular and dorsal root ganglia. However, the major sensory supply to the airways originates from the nodose ganglion. In this study, we evaluated changes in neuropeptide biosynthesis in the sensory airway innervation of ovalbumin-sensitized and -challenged guinea pigs at the mRNA and peptide level. In the airways, a three- to fourfold increase of SP, NKA, and CGRP, was seen 24 h following allergen challenge. Whereas no evidence of local tachykinin biosynthesis was found 12 h after challenge, increased levels of preprotachykinin (PPT)-A mRNA (encoding SP and NKA) were found in nodose ganglia. Quantitative in situ hybridization indicated that this increase could be accounted for by de novo induction of PPT-A mRNA in nodose ganglion neurons. Quantitative immunohistochemistry showed that 24 h after challenge, the number of tachykinin-immunoreactive nodose ganglion neurons had increased by 25%. Their projection to the airways was shown. Changes in other sensory ganglia innervating the airways were not evident. These findings suggest that an induction of sensory neuropeptides in nodose ganglion neurons is crucially involved in the increase of airway hyperreactivity in the late response to allergen challenge.

Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit NOX4 in the Pulmonary Vasculature

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O2) or normoxic (21% O2) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.

The sensory and sympathetic innervation of guinea-pig lung and trachea as studied by retrograde neuronal tracing and double-labelling immunohistochemistry

The sympathetic and sensory innervation of guinea-pig trachea and lung were studied by means of retrograde neuronal tracing using fluorescent dyes, and double-labelling immunofluorescence. Sympathetic neurons supplying the lung were located in stellate ganglia and in thoracic sympathetic chain ganglia T2-T4; those supplying the trachea resided in the superior cervical and stellate ganglia. Retrogradely labelled sympathetic neurons were usually immunoreactive to tyrosine hydroxylase; the majority also contained neuropeptide Y immunoreactivity. However, a small number were non-catecholaminergic (i.e. tyrosine hydroxylase negative), but neuropeptide Y immunoreactive. Within the airways, tyrosine hydroxylase/neuropeptide Y-immunoreactive axons were found in the smooth muscle layer, around blood vessels including the pulmonary artery and vein, and to a lesser extent in the lamina propria. Periarterial axons contained in addition dynorphin immunoreactivity. Sensory neurons supplying the lung were located in jugular and nodose vagal ganglia as well as in upper thoracic dorsal root ganglia; those supplying the trachea were most frequently found bilaterally in the nodose ganglia and less frequently in the jugular ganglia. A spinal origin of tracheal sensory fibres could not be consistently demonstrated. With regard to their immunoreactivity to peptides, three types of sensory neurons projecting to the airways could be distinguished: (i) substance P/dynorphin immunoreactive; (ii) substance P immunoreactive but dynorphin negative; and (iii) negative to all peptides tested. Substance P-immunoreactive neurons innervating the airways invariably contained immunoreactivity to neurokinin A and calcitonin gene-related peptide. Retrogradely labelled neurons located in the nodose ganglia belonged almost exclusively (greater than or equal to 99%) to the peptide-negative group, whereas the three neuron types each represented about one-third of retrogradely labelled neurons in jugular and dorsal root ganglia. Within the airways, axons immunoreactive to substance P/neurokinin A and substance P/calcitonin gene-related peptide were distributed within the respiratory epithelium of trachea and large bronchi, in the lamina propria and smooth muscle from the trachea down to the smallest bronchioli (highest density at the bronchial level), in the alveolar walls, around systemic and pulmonary blood vessels, and within airway ganglia. Those axons also containing dynorphin immunoreactivity were restricted to the lamina propria and smooth muscle. The origin of nerve fibres immunoreactive for vasoactive intestinal polypeptide, of which a part were also neuropeptide Y immunoreactive, could not be determined by retrograde tracing experiments. Vasoactive intestinal polypeptide-immunoreactive fibres terminating within airway ganglia may be of preganglionic parasympathetic origin, whereas others (e.g. those found in smooth muscle) may arise from intrinsic ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea-pig airways

1. The present study addressed the hypothesis that jugular and nodose vagal ganglia contain the somata of functionally and anatomically distinct airway afferent fibres. 2. Anatomical investigations were performed by injecting guinea‐pig airways with the neuronal tracer Fast Blue. The animals were killed 7 days later, and the ganglia were removed and immunostained with antisera against substance P (SP) and neurofilament protein (NF). In the nodose ganglion, NF‐immunoreactive neurones accounted for about 98% of the Fast Blue‐labelled cells while in the jugular ganglion they accounted for approximately 48%. SP and NF immunoreactivity was never (n = 100) observed in the same cell suggesting that the antisera labelled distinct populations. 3. Electrophysiological investigations were performed using an in vitro guinea‐pig tracheal and bronchial preparation with intact afferent vagal pathways, including nodose and jugular ganglia. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in either ganglion. 4. The nodose ganglion contained the somata of mainly fast‐conducting tracheal A delta fibres whereas the jugular ganglion contained equal numbers of C fibre and A delta fibre tracheal afferent somata. The nodose A delta neurones adapted rapidly to mechanical stimulation, had relatively low mechanical thresholds, were not activated by capsaicin and adapted rapidly to a hyperosmotic stimulus. By contrast, jugular A delta and C fibres adapted slowly to mechanical stimulation, were often activated by capsaicin, had higher mechanical thresholds and displayed a slow adaptation to a hyperosmotic stimulus. 5. The anatomical, physiological and pharmacological data provide evidence to support the contention that the vagal ganglionic source of the fibre supplying the airways ultimately dictates its neurochemical and physiological phenotype.

Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart.

Participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig hearts was investigated by applying the NADPH diaphorase technique and immunohistochemistry with NO synthase antiserum. We present evidence that NO synthase is localized in cardiac ganglion cells and nerve fibers innervating the sinuatrial and atrioventricular nodes, the myocardium, local neurons, coronary arteries, and pulmonary vessels, suggesting an involvement of NO in neurogenic heart rate regulation, myocardial cell function, neuronal transmission in cardiac ganglia, and coronary as well as pulmonary vasodilation.

Long-lasting increase of nitric oxide synthase immunoreactivity, NADPH-diaphorase reaction and c-JUN co-expression in rat dorsal root ganglion neurons following sciatic nerve transection

Changes of NADPH-diaphorase reaction (NDP) and nitric oxide synthase immunoreactivity (NOS-IR) in neurons of dorsal root ganglia (DRG) were investigated following transection and ligation of rat sciatic nerve. In untreated rats, 2.7% of L4/L5 DRG neurons were labelled by NDP. After 3 days, intensity of NDP and number of labelled neurons increased and reached a maximal level between 10 and 20 days in 26.8% neurons which persisted up to 50 days. After 150 days, 8.7% of DRG neurons were still labelled. In contralateral L4/L5 DRG, but not L1 and T10 DRG, the number but not the intensity of NDP labelled neurons slightly increased between 10 and 50 days. The patterns of NOS-IR and NDP were congruent. Ipsilaterally, 76% to 92% of NDP neurons showed co-expression with the c-JUN transcription factor which is supposed to play a crucial role in the regeneration process. NDP accumulated in the peripheral nerve stump and was increased in the superficial dorsal horn between 10 and 30 days, whereas motoneurons were not labelled by NOS and NDP.

NOSIP, a novel modulator of endothelial nitric oxide synthase activity

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+‐calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme;however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two‐hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl‐terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin‐1 scaffolding domain. NO production was significantly reduced in eNOS‐expressing CHO cells (CHO‐eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergentinsoluble to the detergent‐soluble fractions of CHO‐eNOS, and this translocation was completelyprevented by transient coexpression of NOSIP in CHO‐eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO‐eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin‐rich membrane fractions of CHO‐eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.—Dedio, J., König, P., Wohlfart, P., Schroeder, C., Kummer, W., Muller‐Esterl, W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J. 15, 79–89 (2001)

Nitric oxide synthase in guinea pig lower airway innervation

The inhibitory non-adrenergic non-cholinergic (i-NANC) innervation of the guinea pig airways was suggested to be mediated, at least partially, by nitric oxide (NO). The enzyme catalyzing the generation of NO and citrulline from L-arginine, nitric oxide synthase (NOS), was found to be identical with neuronal nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase. In the present study, we report the distribution of NOS in guinea pig lower airways and in vagal sensory and sympathetic ganglia as revealed by NOS immunohistochemistry and NADPH-diaphorase histochemistry. The distribution of NOS was identical using either technique and displayed a similar distribution pattern in all parts of the lower airways. Yet, the number of NOS-containing fibres was increasing from cervical trachea towards principal bronchi and decreasing to complete absence in bronchioli. Innervation with NOS-containing nerve fibres was densest in the smooth muscle layer and in the lamina propria of the mucosa. Single fibres were found in the respiratory epithelium. Labelling was absent from nerve fibres innervating the submucosal glands. Perivascular fibre networks enmeshed tracheal arteries, pulmonary arteries and veins. A substantial number of NOS-immunoreactive and NADPH-diaphorase-positive neurons was observed in vagal sensory ganglia, whereas such neurons were rather sparse in sympathetic ganglia. Tracheal and peribronchial ganglia of the airways were devoid of labelling. These findings suggest that extrinsic rather than intrinsic (tracheal and peribronchial) neurons are the source of NO release from guinea pig airway nerve fibres after electrical field stimulation. These extrinsic nerve fibres may originate from both sympathetic and vagal sensory ganglia.

Caveolin-1 Facilitates Mechanosensitive Protein Kinase B (Akt) Signaling In Vitro and In Vivo

Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with &bgr;-cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. &agr;V&bgr;3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of &agr;V&bgr;3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes.

1 The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea‐pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea‐pig cardiac ventricle. 2 A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea‐pig genomic library. 3 The cell‐specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi‐cell RT‐PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. 4 Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. 5 After transfection in human embryonic kidney cells (HEK293) the mean single‐channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. 6 Cell‐attached measurements in isolated guinea‐pig cardiomyocytes (n= 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. 7 Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half‐maximum block at ‐100 mV were 3.24 μm for gpKir2.1, 0.51 μm for gpKir2.2, 10.26 μm for gpKir2.3 and 235 μm for gpKir2.4. 8 Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell‐attached recordings. The concentration and voltage dependence of Ba2+ block of the large‐conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. 9 Our results suggest that the large‐conductance inward rectifier channels found in guinea‐pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate‐conductance (23.8 pS) and low‐conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.