Use of replication-deficient adenoviruses to study signal transduction pathways. Muscarinic responses in HSG and HT29 epithelial cell lines are mediated by G protein βγ-subunits

 HSG and HT29 cells express muscarinic receptors that increase intracellular free Ca²⁺ ([Ca²⁺]_i) by activating phospholipase Cβ. In the present study, we have used the measurement of [Ca²⁺]_i with Fura-2 to show that these receptors are of the M₃ sub-type and that the increase in [Ca²⁺]_i triggered when they are activated is not sensitive to pertussis toxin. We have also used replication-deficient adenoviruses expressing wild-type and dominant-negative mutants of the α-subunits of the heterotrimeric G proteins, G_q and G_i2, to investigate the mechanisms by which these receptors control phospholipase Cβ. We find that the Ca²⁺ response to 100 μmol/l carbachol is not affected by increased expression of the wild-type α-subunit of G_q, but is blocked by the dominant-negative mutant of G_q and by both the wild-type and the dominant-negative mutant α-subunits of G_i2. Expression of α-subunits of G_i2 presumably blocks the response to carbachol by scavenging free βγ-subunits. We conclude that in HSG and HT29 cells, the Ca²⁺ response to M₃ receptor activation is mediated by the βγ- rather than the α-subunits of G_q. Received: 23 September 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999     

Synergistic effects of ATP on oxytocin-induced intracellular Ca2+ response in mouse mammary myoepithelial cells

An increase in intracellular Ca2+ ([Ca2+]i) is essential for mammary myoepithelial cells to contract, leading to milk ejection during lactation. In this study, the intracellular signaling leading to the increase in [Ca2+]i in cultured myoepithelial cells from mouse lactating mammary glands was investigated using fura-2 fluorescence ratiometry. [Ca2+]i increased in cultured myoepithelial cells in response to either oxytocin (1 nM) or ATP (10 microM), and the cells then contracted. These [Ca2+]i responses were diminished by treatment with an inhibitor of phospholipase C (> or = 1 microM U73122). Intracellular application of inositol 1,4,5-trisphosphate (IP3: 10 or 100 microM) increased [Ca2+]i. Pretreatment with pertussis toxin (PTX: 0.1 or 1 microgram/ml) inhibited the [Ca2+]i response to ATP, but had less of an effect on the response to oxytocin. These results indicate that oxytocin and purinergic receptors are coupled to PTX-insensitive and PTX-sensitive G proteins, respectively, and that their activation leads to the increase in [Ca2+]i through the release of Ca2+ from IP3-sensitive intracellular stores via the inositol-phospholipid signaling pathway. Furthermore, we found that the [Ca2+]i responses to oxytocin at physiological doses (0.01-0.1 nM) were augmented in the presence of a sub-responsive dose of ATP (1 microM). The activation of purinergic receptors may facilitate myoepithelial cell contraction in milk-ejection responses.     

Opioid receptor-mediated effects of interleukin-2 on the [Ca2+]i transient and contraction in isolated ventricular myocytes of the rat

Cytokines play significant roles in some cardiovascular disorders, but direct myocardial effects of cytokines remain to be elucidated. In this study, we examined the effects and possible mechanisms of interleukin-2 (IL-2) on contraction and the [Ca2+]i transient of enzymatically isolated ventricular myocytes with spectrofluorometry and video tracking. IL-2 (2.5-200 U/ml) depressed both the contraction and the [Ca2+]i transient in a dose-dependent manner. Pretreatment with the universal opioid receptor antagonist naloxone (10 nM), or a specific kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI, 10 nM), abolished the inhibitory effect of IL-2 on contraction and the [Ca2+]i transient; the specific delta-opioid receptor antagonist naltrindole (1 microM) had no effect. The effect of IL-2 was also abolished after pretreatment with pertussis toxin (PTX, 5 mg/l), but not by genistein (100 microM). Pretreatment with the phospholipase C inhibitor U73122 (5 microM) significantly inhibited the IL-2-induced depression of contraction and the [Ca2+]i transient. It is concluded that the effects of IL-2 on contraction and the [Ca2+]i transient of ventricular myocytes are mediated via the cardiac kappa opioid receptor, and the postreceptor signal transduction pathway includes a PTX-sensitive G protein and phospholipase C.     

Enkephalin induces Ca2+ mobilization in single cells of bradykinin-sensitized differentiated neuroblastoma hybridoma (NG108-15) cells.

A study of the intracellular Ca2+ ([Ca2+]i) response of differentiated neuroblastoma x glioma hybrid cells (NG108-15 cell) to enkephalin (EK) was carried out by fura-2 video-imaging. EK alone did not influence [Ca2+]i in single cells. The opioid did, however, induce a marked [Ca2+]i rise, when the cells were incubated with bradykinin (BK) prior to the EK treatment. Such BK-assisted stimulation of the differentiated hybridoma cells by EK was completely abolished by pertussis toxin treatment. These results suggest that in single NG108-15 cells, EK induces Ca2+ mobilization which is assisted by cross-talk between the EK and BK receptor systems via a pertussis toxin-sensitive G protein.     

Cytopathogenicity of Mycoplasma hyopneumoniae in porcine tracheal ring and lung explant organ cultures alone and in combination with monolayer cultures of fetal lung fibroblasts.

Porcine tracheal rings and lung explants alone and in combination with monolayer cultures of porcine lung fibroblasts (PLF) were separately inoculated with virulent strains of Mycoplasma hyopneumoniae and incubated at various times. The preparations were observed by bright-field, phase-contrast, and scanning electron microscopy. In PLF cultures, the strains at initial concentrations of 10(1.3) colony-forming units/ml increased within 3 days to 10(6) colony-forming units/ml, showed progressive clustering on the cells, and caused some sloughing. Introduction of a tracheal ring or lung explant into these mycoplasma-infected PLF cultures caused the explant to lose its epithelial ciliary motility. Eventually parts or whole cells of the respective ciliated epithelium were lost. Without infected PLF monolayers, the explants inoculated with M. hyopneumoniae were less susceptible to infection. When uninfected explants were incubated for 18 days or kept in stock for 2 months, they did not show the above changes. With 5 h postinoculation, M. hyopneumoniae cultures became intimately associated with the PLF culture, but when epithelial cell sloughing occurred, the mycoplasmal cells became dependent on the introduction of a fresh PLF monolayer or a tracheal or lung explant for survival.     

Identification and characterization of a Mycoplasma hyopneumoniae adhesin.

An adhesin of Mycoplasma hyopneumoniae was identified and characterized in this study. A monoclonal antibody (MAb), F2G5, and its F(ab')2 fragments inhibited the adherence of M. hyopneumoniae to porcine tracheal cilia, the natural targets to which the mycoplasma binds during infection. MAb F2G5 detected multiple bands, but predominantly recognized a 97-kDa (P97) protein of M. hyopneumoniae on immunoblots. Affinity chromatography, conducted with immobilized MAb F2G5, mainly purified P97. The purified proteins were able to bind to cilia and blocked the adherence of intact M. hyopneumoniae cells to cilia. Immunolabeling of mycoplasmas with MAb F2G5 under electron microscopy demonstrated that the proteins recognized by MAb F2G5 were located at the surface of the mycoplasma, predominantly on a surface fuzzy layer. These results indicate that P97 functions as an adhesin of M. hyopneumoniae. The N-terminal amino acid sequence of P97 did not have significant homology with any known bacterial or mycoplasmal adhesins, suggesting that P97 is a novel protein. The predominant proteins detected by MAb F2G5 in different strains varied in size, indicating that the antigen bearing the epitope for MAb F2G5 undergo intraspecies size variation. Antigenic variation of adhesins may be a pathogenic mechanism utilized by M. hyopneumoniae to evade the porcine immune system.     

Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures.

In vivo- and in vitro-grown Mycoplasma hyopneumoniae organisms were inoculated onto newborn piglet tracheal organ cultures to provide a model for interaction of this organism with ciliated respiratory epithelium. Ciliostasis and loss of cilia in tracheal rings were induced by M. hyopneumoniae grown in vivo and with low-passage cultures when grown in vitro. Levels of calmodulin or dehydrogenase enzymes in tracheal ring epithelium were not altered even though ciliostasis and loss of cilia induced by M. hyopneumoniae were extensive. The capacity for inducing epithelial damage diminished with in vitro passage of the organism. Attempts to induce higher-passage cultures to attach to cilia, cause ciliostasis, or cause ciliary damage by supplementation of mycoplasmal medium with porcine lung extract failed. Epithelial damage induced by M. hyopneumoniae in tracheal rings was averted by using porcine immune serum or by separating the organisms from ciliated epithelium with a 0.1-microns-pore-size membrane. Attachment, or at least close association, of M. hyopneumoniae to ciliated epithelium appeared to be necessary to induce ciliostasis and loss of cilia in this model.     

Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors

Previous studies suggest that acetylcholine (ACh) is a transmitter released from taste cells as well as a transmitter in cholinergic efferent neurons innervating taste buds. However, the physiological effects on taste cells have not been established. I examined effects of ACh on taste-receptor cells by monitoring [Ca2+]i. ACh increased [Ca2+]i in both rat and mudpuppy taste cells. Atropine blocked the ACh response, but D-tubocurarine did not. U73122, a phospholipase C inhibitor, and thapsigargin, a Ca2+-ATPase inhibitor that depletes intracellular Ca2+ stores, blocked the ACh response. These results suggest that ACh binds to M1/M3/M5-like subtypes of muscarinic ACh receptors, causing an increase in inositol 1,4,5-trisphosphate and subsequent release of Ca2+ from the intracellular stores. A long incubation with ACh induced a transient response followed by a sustained phase of [Ca2+]i increase. In Ca2+-free solution, the sustained phases disappeared, suggesting that Ca2+ influx is involved in the sustained phase. Depletion of Ca2+ stores by thapsigargin alone induced Ca2+ influx. These findings suggest that Ca2+ store-operated channels may be present in taste cells and that they may participate in the sustained phase of [Ca2+]i increase. Immunocytochemical experiments indicated that the M1 subtype of muscarinic receptors is present in both rat and mudpuppy taste cells.     

ATP acting on P2Y receptors triggers calcium mobilization in primary cultures of rat neurohypophysial astrocytes (pituicytes)

 The effect of adenosine triphosphate (ATP) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of cultured neurohypophysial astrocytes (pituicytes) was studied by fluorescence videomicroscopy. ATP evoked a [Ca²⁺]_i increase, which was dose dependent in the 2.5–50 μM range (EC₅₀=4.3 μM). The ATP-evoked [Ca²⁺]_i rise was not modified during the first minute following the removal of external Ca²⁺. Application of 500 nM thapsigargin inhibited the ATP-dependent [Ca²⁺]_i increase. Caffeine (10 mM) and ryanodine (1 μM) did not affect the ATP-induced [Ca²⁺]_i rise. The pituicytes responded to various P₂ purinoceptor agonists with the following order of potency: ATP=ATP[γ-S]=2-MeSATP≥ADP, where ATP[γ-S] is adenosine 5′-O-(3-thiotriphosphate) and 2-MeSATP is 2-methylthio-adenosine-5′-triphosphate. Adenosine, AMP, α,β-methylene adenosine-5′-triphosphate (α,β-MeATP), β,γ methylene adenosine-5′-triphosphate (β,γ-MeATP) and uridine 5′-triphosphate (UTP) were ineffective. The P₂ purinoceptor antagonists blocked the ATP-evoked [Ca²⁺]_i increase with the following selectivity: RB-2>suramin>PPADS, where RB-2 is Reactive Blue 2 and PPADS is pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The ATP-evoked [Ca²⁺]_i increase was substantially blocked by pertussis toxin treatment, suggesting that it might be mediated by a pertussis-toxin-sensitive G protein. The phospholipase C (PLC) inhibitor U-73122 (0.5 μM) abolished the ATP-evoked [Ca²⁺]_i rise, whereas its inactive stereoisomer U-73343 (0.5 μM) remained ineffective. Our results indicate that, in rat cultured pituicytes, ATP stimulation induces an increase in [Ca²⁺]_i due to PLC-mediated release from intracellular stores through activation of a pertussis-toxin-sensitive, G-protein-linked P_2Y receptor. Received: 24 September 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998     

Ammonium triggers calcium elevation in cultured mouse microglial cells by initiating Ca2+ release from thapsigargin-sensitive intracellular stores

Microglial cells are thought to serve as sensors for pathologic events in the brain. In the present study we demonstrate that these cells respond with an increase in intracellular calcium concentration ([Ca2+]i) to intracellular alkaline shifts induced by either application of NH3/NH4+ or by an extracellular alkaline shift. The cytoplasmic pH (pHi) and [Ca2+]i in cultured mouse microglial cells were studied employing the fluorescent probes BCECF and fura-2, respectively. Application of NH3/NH4+ caused an initial rapid alkalinization followed by a slow recovery towards the resting level, while application of alkaline (pH 8.2) solution triggered a slower rise in pHi. The [Ca2+]i elevation triggered by NH3/NH4+ and extracellular alkaline shift were caused by different mechanisms: extracellular alkalinization induced a transmembrane Ca2+ entry, whereas NH3/NH4+ triggered Ca2+ release from thapsigargin- and ATP-sensitive intracellular pools. The mobilization of intracellular Ca2+ caused by NH3/NH4+ was blocked by a specific inhibitor of phospholipase C, U-73122, but was not affected by an inhibitor of G-protein, pertussis toxin. This implies that NH3/NH4 interacts with phospholipase C and leads to an increase in the intracellular level of inositol 1,4,5-trisphosphate (InsP3). In contrast to a previous study using a microglial cell line, application of NH3/NH4+ did not result in a release of tumor necrosis factor alpha (TNF-alpha), a marker of microglial activation, in the primary microglial cells. This implies that ammonium does not lead to activation of microglia in the culture model.     

Facilitation of calcium influx by propylene glycol through the voltage-dependent calcium channels in PC12 cells

Propylene glycol (PG) raises an intracellular calcium concentration ([Ca2+]i) in PC12 cells. The present study has been undertaken to examine whether or not the voltage-dependent Ca2+ channels are involved in the PG-induced rise in [Ca2+]i and, if so, to determine which types participate in it. CdCl2 (50 micro M) and the Ca2+ -free saline depressed the action of PG (0.5 - 10 %v/v)-induced [Ca2+]i rise. Although NiCl2 (50 micro M) at the same concentration as CdCl2, and omega-agatoxin (50 and 300 nM) had no effect on the PG-induced [Ca2+]i rise, each of omega-conotoxin (1 micro M), nifedipine (10 micro M), nicardipine (10 micro M), varapamil (10 micro M) and diltiazem (10 micro M) significantly decreased it. Electrical stimulation and Bay K 8644 (1 micro M) enhanced the PG-induced [Ca2+]i rise. The second phase of the [Ca2]i rise was fallen fast by nicardipine (10 micro M), but not by omega-conotoxin (1 micro M). The results obtained suggested that the Ca2+ influx through the L- and N-type Ca2+ channels are involved in the PG-induced [Ca2+]i rise.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Platelet activating factor increases ciliary activity in the hamster oviduct through epithelial production of prostaglandin E2

We investigated the signal transduction mechanisms associated with an increase in ciliary beat frequency (CBF) produced by platelet activating factor (PAF) in oviductal ciliated cell cultures. In the range of concentrations similar to that produced by preimplantation embryos, PAF increased the CBF in a dose-dependent manner. The addition of PAF and prostaglandin E2 (PGE2) to the cultures produced a synergic increase of ciliary beating, suggesting that PAF and PGE2 signal transduction pathways may be associated. To demonstrate this hypothesis, cyclooxygenase-2 (COX-2) was selectively blocked by a specific inhibitor, NS-398, and the PAF-induced CBF increase was abolished. Moreover, a phospholipase A2 (PLA2) inhibitor, AACOCF3, blocks the PAF-induced CBF increase. PGE2 production by oviductal epithelial cells is stimulated by PAF, and WEB-2086, a PAF-receptor blocker, specifically blocks the PAF-induced PGE2 production. Using the fluorescent indicator fura-2, we measured the effect of PAF on intracellular Ca2+ concentration ([Ca2+]i) in individual ciliated cells. PAF induced a transient increase of [Ca2+]i that was blocked by WEB-2086 or by removal of extracellular Ca2+. We propose a mechanism for PAF-mediated signal transduction in the ciliated cells of the oviductal epithelium. Minimal doses of PAF trigger Ca2+ mobilization in tandem with increased PLA2 activity and a COX-2-mediated increase in PGE2. Local PGE2 production by the oviductal mucosa suggests the presence of an autocrine loop controlling ciliary activity.     

The morphological and functional observation of the gap junction proteins in the oviduct epithelia in young and adult hamsters

The histological morphology of oviduct epithelia have been described well, however, the expression pattern of the gap junction proteins in the cells, and the function related with the proteins, such as [Ca2+]i dynamics pattern of living oviduct epithelia at different ages have not been clarified. We used immunohistochemistry to compare the expression pattern of gap junction proteins in the cells of the young and adult groups. Moreover, we used real-time confocal microscopy to observe the spontaneous Ca2+ oscillation (spontaneous fluctuation) in freshly isolated epithelia (ciliated cells) in ampulla potion of oviduct from the two groups. The results show as demonstrated by immunohistochemistry the gap junction proteins (Cx26, Cx32 and Cx43) formed a well-regulated expression in the young animals, but not in the adult animals. In addition, the [Ca2+]i dynamics of ciliated cells in freshly oviduct epithelia have a spontaneous fluctuation pattern that occurs without any stimulation in the young animals, but this pattern was not observed in the adult animals. In conclusions, our findings suggest that gap junctions regulate the spontaneous fluctuation of [Ca2+]i dynamics in ciliated cells of oviduct epithelia in young animals.     

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.     

Evidence for P2Y-type ATP receptors on the serosal membrane of frog skin epithelium

The present study presents the first evidence for P2Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca2+]i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca2+ in the solutions ATP increased [Ca2+]i. The increase in [Ca2+]i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca2+]i caused by the various compounds. The rise in [Ca2+]i was predominantly monophasic at low ATP concentrations (below 100 microM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca2+ in the extracellular solution ATP caused Ca2+ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca2+]i although the plateau level of the [Ca2+]i response was higher with this P2Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P2Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.     

Relationship of free intracellular calcium to the cytolytic activity of Entamoeba histolytica.

Entamoeba histolytica adherence and destruction of host cells is required for in vivo pathogenicity; amebic in vitro adherence is mediated by a galactose- or N-acetyl-D-galactosamine-inhibitable surface lectin (Gal/GalNAc adherence lectin). Free intracellular Ca2+ concentration [( Ca2+]i) was measured in living amebae and target cells during amebic cytolysis of Chinese hamster ovary (CHO) cells and human polymorphonuclear neutrophils by utilizing the Ca2+ probe Fura-2 and computer-enhanced digitized microscopy. Motile E. histolytica trophozoites had oscillatory increases in [Ca2+]i in head or tail regions; however, there was no increase in regional or total amebic [Ca2+]i upon contact with a target CHO cell. Target CHO cells and polymorphonuclear neutrophils demonstrated marked irreversible increases in [Ca2+]i within 30 to 300 s following contact by an ameba (P less than 0.01); increased [Ca2+]i preceded the occurrence of nonspecific surface membrane permeability and death of the target cell. Target CHO cells contiguous on a monolayer to a cell contacted by an ameba experienced a rapid but reversible rise in [Ca2+]i (P less than 0.01) and were not killed. Galactose (40 mg/ml) totally abrogated the rise in target CHO cell [Ca2+]i that followed contact by amebae (P less than 0.01); immunoaffinity-purified amebic Gal/GalNAc adherence lectin (0.25 micrograms/ml) induced a rapid and reversible rise in CHO cell [Ca2+]i (P less than 0.01) which was inhibited by galactose. Amebic [Ca2+]i was not elevated following parasite adherence to target cells; a rapid and substantial rise in target cell [Ca2+]i occurred which was mediated, at least in part, by the Gal/GalNAc adherence lectin of the parasite and led to the death of target cells.     

Microtiter plate adherence assay and receptor analogs for Mycoplasma hyopneumoniae.

A microtiter plate adherence assay for Mycoplasma hyopneumoniae was established by use of purified swine tracheal cilia which contained receptors for the mycoplasmas. M. hyopneumoniae bound specifically to plates coated with solubilized cilia. The binding was dependent on both the concentration of cilia and the number of mycoplasmas. Dextran sulfate, heparin, chondroitin sulfate, laminin, mucin, and fucoidan significantly inhibited the binding of the mycoplasmas. The six inhibitors also disrupted the adherence of the mycoplasmas to intact ciliated cells. Preincubation with either mycoplasmas or cilia indicated that heparin, mucin, fucoidan, and chondroitin sulfate interacted with the adhesive molecules on the surface of the mycoplasmas, while laminin blocked the receptors in cilia. The basis for the inhibition induced by dextran sulfate was unknown. Treatment of cilia with neuraminidase appeared to promote adherence of the mycoplasmas, whereas treatment of cilia with sodium metaperiodate decreased binding. These results indicate that receptors for M. hyopneumoniae in the ciliated epithelium of the respiratory tract of pigs are glycoconjugate in nature.     

Alpha-adrenoceptor agonists produce Ca2+ oscillations in isolated rat aorta: role of protein kinase C.

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) in responses to norepinephrine (NE) and selective alpha2-adrenoceptor agonist, UK14,304 of the endothelium-denuded rat aorta loaded with fura PE-3. NE (3 x 10(-8) M) evoked a rapid increase in [Ca2+]i followed by slight decreasing to a steady state level and produced a contraction. After the NE-induced increase in [Ca2+]i had reached a maximum, the [Ca2+]i showed persistent oscillations. The Ca2+ oscillations were superimposed on the sustained increase in [Ca2+]i. UK14,304 (3 x 10(-6) M) also evoked an increase in [Ca2+]i and produced a contraction. However, the UK14,304-induced effect on [Ca2+]i was characterized by pronounced oscillations, and the amplitude of the sustained increase in [Ca2+]i was less than that seen with NE. Protein kinase C inhibitor, Ro31-8220 (3 x 10(-6) M) and verapamil (10(-5) M) abolished both NE and UK14,304-evoked Ca2+ oscillations. UK14,304-induced contractions were also strongly inhibited by Ro31-8220 and verapamil. However, NE induced contractions were partly inhibited by these inhibitors. The sustained increases in [Ca2+]i evoked NE and UK14,304 were not significantly inhibited by Ro31-8220 and verapamil. These results suggest that NE and UK14,304 produce Ca2+ oscillations during sustained contractions in rat aorta. The alpha2 adrenoceptor agonist, UK14,304-induced sustained contraction and Ca2+ oscillations may be due to PKC activation and opening of voltage-dependent L type Ca2+ channels.