Mycoplasma pneumoniae induces airway epithelial cell expression of MUC5AC in asthma.

As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects. A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air-liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-kappaB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA. M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-kappaB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups. Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.

Liquid transport properties of porcine tracheal epithelium.

Because of its possible importance to the etiology of cystic fibrosis lung disease, the ion and water transport properties of tracheal epithelium were studied. Net liquid flux (J(V)) across porcine tracheal epithelium was measured in vitro using blue dextran as a volume probe. Luminal instillation of isosmotic sucrose solution (280 mM) induced a small net secretion of liquid (7.0 +/- 1.7 nl x cm(-2) x s(-1)), whereas luminal hyposmotic sucrose solutions (220 or 100 mM) induced substantial and significant (P < 0.05) liquid absorption (34.5 +/- 12 and 38.1 +/- 7.3 nl x cm(-2) x s(-1), respectively). When the luminal solution was normal (isosmotic) Krebs buffer, liquid was absorbed at 10.2 +/- 1.1 nl x cm(-2) x s(-1). Absorptive J(V) was abolished by 100 microM amiloride in the luminal solution and significantly reduced when the luminal solution was Na(+)-free Krebs solution. Absorptive J(V) was not significantly affected by 300 microM 5-nitro-2-(3-phenylpropylamino)benzoate or 100 microM diphenylamine-2-carboxylic acid, both cystic fibrosis transmembrane conductance regulator protein (CFTR) inhibitors, in the instillate but was significantly reduced by 60% when the luminal solution was Cl(-)-free Krebs solution. We conclude that water freely permeates porcine tracheal epithelium and that absorption of liquid is normally driven by active transcellular Na(+) transport and does not require the CFTR.

C-Terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit.

The calcium channel alpha(1A) subunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The alpha(1A) C terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of alpha(1A) subunits. Specifically, the biophysical properties of two alternatively spliced alpha(1A) subunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression in Xenopus oocytes demonstrated that the two variants were similar with respect to current-voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which alpha(1A) subunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca(2+) channel function.

CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands.

Previous studies demonstrated that ACh-induced liquid secretion by porcine bronchi is driven by active Cl(-) and HCO(-)(3) secretion. The present study was undertaken to determine whether this process was localized to submucosal glands and mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). When excised, cannulated, and treated with ACh, porcine bronchi secreted 15.6 +/- 0.6 microliter. cm(-2). h(-1). Removal of the surface epithelium did not significantly affect the rate of secretion, indicating that the source of the liquid was the submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a relatively nonselective Cl(-)-channel blocker, significantly reduced liquid secretion by 86%, whereas pretreatment with DIDS, which inhibits a variety of Cl(-) channels but not CFTR, had no effect. When bronchi were pretreated with glibenclamide or 5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR), the rate of ACh-induced liquid secretion was significantly reduced by 39 and 91%, respectively, compared with controls. Agents that blocked liquid secretion also caused disproportionate reductions in HCO(-)(3) secretion. Polyclonal antibodies to the CFTR bound preferentially to submucosal gland ducts and the surface epithelium, suggesting that this channel was localized to these sites. These data suggest that ACh-induced gland liquid secretion by porcine bronchi is driven by active secretion of both Cl(-) and HCO(-)(3) and is mediated by the CFTR.

Pressor reflex evoked by static muscle contraction: role of nitric oxide in the dorsal horn.

In this study, we tested the hypothesis that nitric oxide (NO) production in the dorsal horn is involved in producing the pressor reflex elicited by static contraction of skeletal muscle. Cats were anesthetized with alpha-chloralose (80 mg/kg) and urethane (100 mg/kg), and a laminectomy was performed. With the exception of the L7 dorsal root, the dorsal and ventral roots from L5 to S2 were sectioned on one side and static contraction of the ipsilateral triceps surae muscle was evoked by electrically stimulating the peripheral ends of the L7 and S1 ventral roots. Dialysis of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 50 mmol/l syringe concentration, based upon dose-response data) into the dorsal horn at L6 and S1 failed to attenuate the peak change in mean arterial pressure (MAP) evoked by static contraction (DeltaMAP in mmHg: 57 +/- 5 before and 50 +/- 6 after 2 h of L-NAME). However, this dialysis of L-NAME reduced the magnitude of the initial pressor response as the MAP at 10 s of the contraction fell from 27 +/- 4 to 17 +/- 4 mmHg. On the other hand, 2 h of L-arginine dialysis (50 mmol/l) shifted the curve representing the time course of the pressor response upward and increased the peak pressor response to static contraction from 51 +/- 9 to 68 +/- 9 mmHg. A 2-h dialysis of D-NAME (50 mmol/l), the inactive enantiomer of L-NAME, had no effect on the time course or the peak pressor response (DeltaMAP in mmHg: 78 +/- 12 before and 72 +/- 15 after). These data suggest that NO production in the dorsal horn has a modulatory influence on the pressor reflex evoked by static contraction of skeletal muscle and that increasing the level of NO in the dorsal horn enhances the excitability of dorsal horn cells to muscle afferent input.

Segmental effect of NMDA block in the dorsal horn on the pressor reflex.

The purpose of this study was to examine the influence of NMDA receptor blockade in the dorsal horn of adjacent spinal segments as it pertains to the pressor reflex evoked by static contraction and stretch of skeletal muscle. In this preparation, cats were anesthetized and the afferent fibers mediating the pressor reflex entered the spinal cord via the L7 dorsal root. Blockade of dorsal horn NMDA receptors at L6 and L7 attenuated the pressor reflex evoked by static contraction and muscle stretch. However, NMDA block in the L6 dorsal horn alone failed to alter the peak increase in MAP produced by static contraction and muscle stretch, but the initial pressor response evoked by static contraction was attenuated. These data support the hypothesis that the pressor reflex is partially mediated by activation of NMDA receptors in the dorsal horn, and this occurs at multiple spinal segments. Further, these data suggest that activation of NMDA receptors plays an important role in initiating the rise in arterial pressure produced by static contraction of skeletal muscle.

Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor.

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2+]i and NO release due to shear stress or agonist (ATP or brominated Ca2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 microL). The microsensor was positioned approximately 100 microns above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm2. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2+]i, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133 +/- 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 +/- 2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 mumol/L) or Br-A23187 (10 mumol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 mumol/L) chelation of extracellular Ca2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 +/- 0.07 nmol/L per second) closely resembled the kinetics of Ca2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64 +/- 0.32 nmol/L per second). These data argue that shear stress causes a Ca(2+)-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel beta subunit increases rate of inactivation of specific voltage-gated potassium channel alpha subunits.

Voltage-gated potassium channel beta subunits are cytoplasmic proteins that co-purify with the pore-forming alpha subunits. One of these subunits, Kv beta 1 from rat brain, was previously demonstrated to increase the rate of inactivation of Kv1.1 and Kv1.4 when co-expressed in Xenopus oocytes. We have cloned and characterized a novel voltage-gated K+ channel beta subunit. The cDNA, designated Kv beta 3, has a 408-amino acid open reading frame. It possesses a unique 79-amino acid N-terminal leader, but is identical with rat Kv beta 1 over the 329 C-terminal amino acids. The Kv beta 3 transcript was found in many tissues, but was most abundant in aorta and left ventricle of the heart. Co-expression of Kv beta 3 with K+ channel alpha subunits shows that this beta subunit can increase the rate of inactivation from 4- to 7-fold in a Kv1.4 or Shaker B channel. Kv beta 3 had no effect on Kv1.1, unlike Kv beta 1 which can increase rate of inactivation of this alpha subunit more than 100-fold. Other kinetic parameters were unaffected. This study shows that voltage-gated K+ channel beta subunits are present outside the central nervous system, and that at least one member of this family selectively modulates inactivation of K+ channel alpha subunits.

Coordinate depression of bradykinin receptor recycling and microtubule-dependent transport by taxol.

Significant cardiovascular side effects have limited the use of taxol as an anticancer drug. A link between decreased plasma membrane dynamics and taxol has been implied because taxol can inhibit intracellular vesicle movements. Reduced membrane recycling caused by taxol could inhibit agonist-evoked Ca2+ signaling within endothelial cells, resulting in endothelium-dependent vasodilation. Bradykinin and ATP are two agonists that evoke Ca2+ transients in endothelial cells. Since the bradykinin receptor-agonist complex is internalized and recycled whereas the ATP agonist-receptor complex is not, we expected that a taxol inhibition of recycling would decrease bradykinin but not ATP receptor activity. We found that taxol depresses (i) the frequency (to 41% of control) and velocity (to 55% of control) of microtubule-dependent vesicle transport and (ii) bradykinin-evoked cytosolic Ca2+ transients (to 76% of control) in bovine aortic endothelial cells. In studying bradykinin receptor desensitization, which reflects receptor recycling, we demonstrate that taxol inhibits bradykinin-evoked Ca2+ transients by 50%. Taxol did not significantly alter ATP-evoked Ca2+ transients in either single-exposure or desensitization experiments. We suggest that taxol's reduction of bradykinin-evoked Ca2+ transients is due to altered microtubule-dependent membrane recycling. This report describes taxol's ability to alter plasma membrane composition through effects on vesicle transport and membrane trafficking pathways. This finding provides a possible mechanism by which taxol can substantially alter cardiovascular function.

Voltage and temperature dependence of single K+ channels isolated from canine cardiac sarcoplasmic reticulum.

The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive. Nonlinear least-squares analysis of dwell times revealed that the closed state was biexponential and was thus composed of a fast (Cf) and a slow (C8) component.Tcf was voltage insensitive with an average value of 5.9 ms, whereas tau c was approximately two orders of magnitude slower and was voltage dependent. The voltage dependence of Cs was described by Tau c (ms) = exp(-0.025-(Vm (mV) - 250)).

Schistosoma mansoni: influence of infection on levels of translatable mRNA and on polypeptide synthesis in the ovotestis and albumen gland of Biomphalaria glabrata.

Larval trematode infection causes a disruption of normal reproductive activity in the molluscan intermediate host. Because relatively little is known about the dynamics of this host-parasite interaction, the effect of Schistosoma mansoni infection on translatable mRNA pools and on polypeptide synthesis was examined in the ovotestis (OT) and albumen gland (AG) of Biomphalaria glabrata. Total RNA was isolated from OTs and AGs from uninfected control snails and snails at 14, 21, and 28 days postinfection (pi) with 20 S. mansoni miracidia and subjected to a rabbit reticulocyte in vitro translation system. Quantitative densitometry of autofluorograms of one-dimensional SDS-PAGE slab gels revealed reductions in quantities of total proteins synthesized in vitro from RNA isolated from infected OTs at 21 and 28 days pi, but not at Day 14 pi. Similar reductions were seen in 10 individual polypeptides selected for a more detailed analysis. In contrast to the OT, Day 14 pi-infected AGs exhibited an initial increase in total protein synthesized in the in vitro translation system utilized, followed by significant reductions at 21 and 28 days pi. Selective modulation of labeled polypeptides was evident in 11 polypeptides chosen for a more detailed analysis. This general pattern of parasite inhibitory effects was also seen in parallel pulse-chase studies using [35S]methionine metabolic labeling of in vitro-cultured OTs and AGs. In these experiments, significant reductions in the amounts of labeled polypeptides found in culture supernatants at 14, 21, and 28 days pi were evident. Total polypeptide synthesis also was solubilized AGs from infected snails at 21 and 28 days pi. Results indicate that larval trematode infection induced a generalized disruption of polypeptide metabolism in OTs and AGs of B. glabrata. Such inhibition may occur at both the transcriptional and the translational levels and is initially manifested early in infection, during the time that daughter sporocysts begin to migrate and colonize the digestive gland and OT.

Influence of larval schistosomes on polysaccharide synthesis in albumin glands of Biomphalaria glabrata.

An in vitro bioassay was used to examine [14C]glucose incorporation into polysaccharides in albumen glands (AGs) of susceptible M-line Biomphalaria glabrata infected with the NMRI strain of Schistosoma mansoni. Polysaccharide and galactogen synthesis were unaffected by larval trematode infection in AGs of snails at days 14, 21, and 28 post-infection (p.i.) when compared to uninfected controls. Further experiments were conducted to determine if daughter sporocysts, hypothesized to be primary mediators of parasitic castration in this system, were able to exert direct effects on synthetic activity of uninfected AGs via haemolymph-borne molecules or in vitro culture-generated larval excretory-secretory (ES) products. When AGs were incubated in the presence of infected snail haemolymph, significant differences in quantities of polysaccharides and galactogen were detected only in test organs incubated in day 28 p.i. haemolymph. Daughter sporocyst ES products generated during the first 48 h of culture caused a significant reduction in polysaccharide and galactogen synthesis in test organs. When ES products from days 3 to 6 of in vitro culture were tested similarly, no significant differences in either polysaccharide or galactogen synthesis were observed between control and test organs. These data demonstrate that daughter sporocysts are able to modulate a specific aspect of the reproductive activity of the snail host through haemolymph-borne molecules of host or parasite origin, or directly through in vitro culture-generated ES products.

Schistosoma mansoni: effect of infection on reproduction and gonadal growth in Biomphalaria glabrata.

Sexually mature Biomphalaria glabrata were exposed to 12 miracidia of Schistosoma mansoni, and egg production of snails was monitored over a period of 5 weeks. During the study period, exposed snails grew at approximately the same rate as unexposed controls. Castration, as measured by a reduction in the mean number of eggs laid per snail occurred between 14 and 21 days postexposure (PE). The reduction in fecundity in infected snails coincided with the migration and establishment of daughter sporocysts in the digestive gland and gonad. Enumeration of individual oocytes in longitudinal sections of the ovotestis revealed that uninfected snails contained significantly more oocytes per section than infected snails at 27, 31, and 40 days PE. In addition, the mean area of gonadal sections of control snails increased over the 40-day experimental period, whereas there was no such increase in gonadal area of infected snails. These data suggest that there is an inhibition in gonadal growth in infected snails. When oocyte data were expressed in terms of mean gonadal area, the mean number of oocytes per mm2 of gonad of uninfected and infected snails did not differ significantly over the study period, except at Day 14 PE, when infected snails contained a significantly greater number of oocytes per mm2 of gonad than did uninfected controls. It is hypothesized that daughter sporocysts of S. mansoni are primarily responsible for the inhibition of host reproductive activity, and may be mediating their effects through mechanisms involved in the regulation of gonadal growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of guinea pig myometrial beta-adrenoreceptors by guanosine triphosphate and magnesium ion.

The effects of guanosine triphosphate (GTP) and magnesium on the interaction of 1-isoproterenol with beta-adrenoreceptors were studied in myometrial membranes from nonpregnant and late-pregnant (0.9 gestation, term 65 days) guinea pigs. The affinity of the beta-adrenoreceptor for 1-isoproterenol, as measured by inhibition of (-)125I-cyanopindolol binding, was increased by 10 mM MgCl2. The addition of 250 microM GTP reversed this process. In the presence of MgCl2, the competition curves could be resolved into two affinity states of the beta-adrenoreceptor, high and low, respectively. The ratio of the dissociation constant of the high-affinity state to that of the low-affinity state was significantly higher in late-pregnant than in the nonpregnant animals. In the presence of GTP, there was only one (low-affinity) state of the receptor detectable. In both groups of animals, the interaction between beta-adrenoreceptor agonists and myometrial beta-adrenoreceptors was positively modulated by MgCl2. This process was reversed by GTP. However, there appeared to be a differential regulation of the ability of the myometrial beta-adrenoreceptor to form a high-affinity state, depending on the reproductive state of the animal.

Up-regulation of guinea pig myometrial beta-adrenoreceptors by systemic estradiol and progesterone.

The effects of systemic administration of 17 beta-estradiol (E2) and progesterone (P) on the concentration and affinity of myometrial beta-adrenoreceptors were studied in non-pregnant, previously oophorectomized guinea pigs receiving a continuous infusion of E2, P, a combination of the two hormones, or placebo for 7 days. Myometrial beta-adrenoreceptors were characterized by using (-)-[125I-cyanopindolol as the specific beta-adrenoreceptor ligand. Compared to that in the control group, E2 administration resulted in a 7-fold increase in the density of myometrial beta-adrenoreceptors. Administration of P alone resulted in a significant increase in both beta-adrenergic receptor concentration and the receptor dissociation constant (Kd). Finally, a combination of E2 and P treatment did not result in any synergistic or additive effect for the beta-adrenergic receptors, while the Kd was twice that of the control or E2-treated animals. We conclude that systemic administration of these sex steroid hormones, directly or indirectly, modulates myometrial beta-adrenergic receptor concentrations and their ligand affinity.

Genotype frequency differences in Halipegus occidualis-infected and uninfected Helisoma anceps.

Allozyme frequencies in Helisoma anceps infected with the hemiurid trematode, Halipegus occidualis, were compared with those of uninfected H. anceps from a small, North Carolina farm pond. Of 6 loci found to be polymorphic, the frequencies of esterase-1 and leucine aminopeptidase were different in infected and uninfected snails. Genetic heterozygosity, as determined by starch gel electrophoresis, was greater in uninfected H. anceps relative to infected individuals. These observations combined with the high prevalence (up to 60%), complete castration in patent infections, and the absence of an encapsulation response in infected snails, suggest that factors conferring incompatibility may have been selected for in the H. anceps population within the pond.

Protein synthesis and transport by the rat choroid plexus and ependyma: an autoradiographic study.

Light (LM-ARG) and electron microscope (EM-ARG) autoradiographs were preapred from immature rat choriod plexus and ependyma at 5, 10, 30, and 60 min and 16 h following intraperitoneal administration of [3H]labeled amino acid mixtures. Intracellular protein synthesis and transport were ascertained in lateral and fourth ventricle choroid plexus epithelium by quantitative EN-ARG at the several post-injection intervals. ARG were also prepared from choriod plexuses cultured for one day, pulse labeled for one hour and reincubated for various periods in nonradioactive media. Significant labeling of both attached and free apical protrusions (blebs) was observed in both choroid plexus and ependyma in vivo and in choroid plexus in vitro. This phenomenon was interpreted as a physiologically significant mechanism for protein trasport (apocrine secretion) by epithelia into the cerebrospinal fluid (CSF).