Theophylline and cAMP inhibit lysophosphatidic acid-induced hyperresponsiveness of bovine tracheal smooth muscle cells

We have established an in vitro model of airway hyperresponsiveness, using a bovine tracheal smooth muscle cell (BTSMC)-embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates the small G protein RhoA, ATP- and high K⁺ solution-induced gel contraction was significantly augmented. This was not due to the modulation of Ca²⁺ mobilizing properties, since ATP- and high K⁺-induced Ca²⁺ transients were not significantly different between control and LPA-treated BTSMC. Y-27632, an inhibitor of Rho-kinase, suppressed the LPA-induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (> 1 μ m ) reversed the LPA-induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 μ m ). We confirmed that theophylline increased the intracellular concentration of cAMP ([cAMP]_i) in BTSMC. Elevation of [cAMP]_i with dibutyryl cAMP or forskolin also reversed the LPA-induced augmentation of gel contraction. Furthermore, theophylline, as well as dibutyryl cAMP and forskolin, suppressed the LPA-induced membrane translocation of RhoA, indicating that they prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA-induced, RhoA/Rho-kinase-mediated hyperresponsiveness of tracheal smooth muscle cells due to the accumulation of cAMP.     

Excitation and contraction in bovine tracheal smooth muscle.

1. The smooth muscle layer of the bovine trachea was studied in vitro with the micro-electrode and sucrose-gap techniques. The membrane potential was stable at--47-6 plus or minus 0-98 (S.E. of mean) mV, and there was no spontaneous electrical or mechanical activity. 2. The cell membrane had strong rectifying properties, making it impossible to elicit action potentials by electrical stimulation in normal Krebs Solution. The rectification was abolished by TEA (30 mmol/l), which depolarized the membrane and produced plateau-type action potentials. 3. The spontaneous repetitive action potentials produced by TEA were associated with rhythmic oscillatory contractions of the muscle strips. 4. Histamine caused an increased tone, with superimposed rhythmic fluctuations in tension. The electrical response consisted of depolarization, with rhythmic slow oscillations in potential (slow waves) which were synchronous with the fluctuations in tension. 5. Acetylcholine produced smooth, tonic contractures of tracheal muscle strips, and caused simple depolarization of the membrane. No action potentials were recorded. 6. In calcium-free solutions containing EGTA, the mechanical response to TEA was completely abolished; the response to histamine was greatly reduced; the response to acetylcholine was reduced to a lesser extent. All responses reverted to normal when normal concentrations of extracellular calcium were restored. 7. Lanthanum added to the bathing solution abolished the contraction due to TEA even though the solution contained calcium. It reduced the histamine-induced contraction to 26% of control, and reduced the acetylcholine-induced contraction to 58% of control; extracellular calcium was present throughout. 8. It is suggested that TEA produces contraction by promoting influx of calcium ions into the cytoplasm. Acetylcholine, and to a smaller extent histamine, are less dependent upon the presence of extracellular calcium, and may be capable of releasing calcium sequestered within the cell; acetylcholine appears to be more effective in releasing sequestered calcium.     

Antiviral activity in interferon-treated bovine tracheal organ cultures.

The effect of bovine interferon on the replication of infectious bovine rhinotracheitis virus and vesicular stomatitis virus in bovine tracheal organ cultures was studied. After treatment of tracheal organ cultures with interferon, inhibition of infectious bovine rhinotracheitis virus and vesicular stomatitis virus replication was observed. This tracheal organ system may be useful in determining the in vivo response to interferon for viral infections of the bovine respiratory tract.     

cAMP microdomains and L-type Ca2+ channel regulation in guinea-pig ventricular myocytes

Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by β-adrenergic receptor (βAR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP-dependent stimulation of the L-type Ca²⁺ current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP₄ prostaglandin receptors, which are known to activate G_s-dependent signalling pathways. Using fluorescence resonance energy transfer-based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor-mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a G_i-dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with βAR regulation of L-type Ca²⁺ channel function.     

Suppression of maxi-K channel and membrane depolarization by synthetic polycations in single tracheal myocytes

Polycationic proteins, e.g., major basic protein from eosinophils or cathepsin G from neutrophils, have been shown to increase nonspecific airway responsiveness. Along with several indirect manners of action, polycations were reported to contract smooth-muscle strips and to raise the cellular Ca(2+) concentration as a direct action on airway smooth muscle. However, the mechanistic basis for the direct behavior remains to be elucidated. To address this issue, we examined the effects of synthetic cationic polypeptides poly-L-arginine and poly-L-lysine on fresh single smooth-muscle cells from bovine trachea using a patch-clamp technique. Both of the polycations significantly depolarized the membrane from a baseline of about -40 to -20 mV in a dose-dependent manner. The polycations also suppressed whole-cell spontaneous transient outward currents as well as both the conductance (from a baseline of about 130 to 70 pS) and open-state probability (about 25% of control values) of large-conductance Ca(2+)-dependent K(+) channel (maxi-K channel) on excised outside-out patch membranes. The polycations were without effect on the whole-cell Ca(2+) currents induced by depolarizing voltage pulses. We concluded that the synthetic polycations had at least two sites of action; one is the delayed rectifier K(+) channel that is responsible for the membrane depolarization that increases Ca(2+) influx, and the other is the maxi-K channel the suppression of which inhibits muscle relaxation. These results may explain the direct contractile action and, therefore, one of the mechanisms underlying the airway hyperresponsiveness induced by various polycationic proteins.     

Cell-volume regulation by swelling-activated chloride current in guinea-pig ventricular myocytes

The cell-volume regulation by swelling-activated Cl- current (I(Cl,swell)) was studied in guinea pig ventricular myocytes, using a microscopic video-image analysis. We have previously shown that in ventricular cells depolarized in high-K+ ([K+]o>45 mM) solution, an activation of the cyclic AMP-dependent Cl- current (I(Cl,cAMP)) leads to cell swelling. We first investigated the mechanism underlying the I(Cl,cAMP)-independent recovery (shrinkage) of the swollen cells. They shrank when the membrane potential (Vm) was made negative to the equilibrium potential of Cl- (ECl) by lowering [K+]o or [Cl-]o in the high-K+ solution. This shrinkage was attenuated by the inhibitors (DIDS, glibenclamide, furosemide) of swelling-activated Cl- current (I(Cl,swell)). These findings suggested an involvement of I(Cl,swell) in the observed isosmotic cell shrinkage. On the other hand, an application of hyposmotic (70% of control) solution to the cells at normal [K+]o (ECl>Vm) induced a cell swelling, and the swollen cells underwent a slight but definite spontaneous cell shrinkage during hyposmotic challenge, indicating the operation of the mechanism of regulatory volume decrease (RVD). This RVD was pronounced at low [Cl-]o, at which ECl was much more positive than Vm. On the contrary, when the hyposmotic solution was applied to the cells at high [K+]o, at which ECl was negative to Vm, the cells swelled vigorously and monotonically without showing RVD, the swelling being much greater than that seen at normal [K+]o. Both the RVD at normal [K+]o and the extra cell swelling at high [K+]o were suppressed by DIDS. These results suggest that I(Cl,swell) activated by cell swelling can shrink or inflate the cardiac cells under hyposmotic as well as isosmotic conditions, depending on Vm and ECl.     

Amiloride antagonizes beta-adrenergic stimulation of cAMP synthesis and Cl- secretion in human tracheal epithelial cells.

Amiloride, a potent blocker of the sodium channel in airway epithelium, has been administered by aerosol as a therapeutic agent for cystic fibrosis. Because amiloride in high concentration has been reported to interfere with cell functions, including adrenergic responses, we tested the ability of amiloride to inhibit beta-adrenergic responses in human tracheal epithelial cells. Amiloride (10(-4) M), applied from the basolateral surface of a cell monolayer, inhibited the changes in transepithelial potential and short circuit current to isoproterenol (10(-6) M). The stimulation of cyclic adenosine monophosphate (cAMP) synthesis by isoproterenol was inhibited in dose-dependent fashion by amiloride (P = 0.007 by multivariate ANOVA with multiple samples correction). Amiloride did not affect baseline transepithelial potential, short circuit current, basal cAMP levels, cAMP response to prostaglandin E2, or basal adenylate cyclase activity measured directly in membrane preparations. Therefore, it is unlikely that amiloride exerts a nonspecific toxic effect on adenylate cyclase, receptor-cyclase coupling, or substrate or cofactor supply. The binding of [125I]iodocyanopindolol (ICYP), a beta-adrenergic receptor antagonist, to membranes from human tracheal epithelial cells could be displaced by amiloride with IC50 = 410 microM; displacement was 70% at 10(-3) M amiloride. These data are most consistent with the hypothesis that amiloride inhibits beta-adrenergic responses in airway epithelial cells by occupying beta-adrenergic receptor sites. Therapeutic administration of amiloride should take into account its affinity for adrenergic receptors.     

Non-specific inhibition by virus particles of human lymphocytes mitogenesis.

Many different types of virus particles including avian retroviruses, Friend leukaemia virus and Sendai virus are able, when coincubated with human peripheral blood lymphocytes in the presence of mitogens or alloantigens, to inhibit the usual proliferative responses that normally ensue. These effects are independent of infection and can be obtained using u.v.-inactivated viruses as well as virus-lymphocyte combinations which are non-physiological in nature. Lymphocytes which are preincubated with viruses for as little as 5 min, and then washed free of unbound virus, are significantly impaired in terms of ability to react to mitogenic stimulus. These events may be mediated, in part at least, by the virus-induced elaboration by mononuclear cells of a factor with lymphocyte inhibitory potential.     

Actions of pinacidil on membrane currents in canine ventricular myocytes and their modulation by intracellular ATP and cAMP

We studied the effects of pinacidil (3–50 M) on the membrane currents of canine ventricular myocytes, using the whole-cell variant of the patch-clamp technique, and the modulation of these effects by intracellular environment, using the pipette perfusion technique. The following observations were obtained: (1) pinacidil induced a dosedependent outward shift in current at voltages positive to ±70 mV; (2) the pinacidil-induced current was largely timeindependent at voltages positive to ±50 mV and displayed an increase in current fluctuations at more positive voltages, resembling the kinetic properties of current through the ATP-regulated K⁺ channels; (3) elevating the extracellular potassium concentration ([K⁺]_o) caused a positive shift in the voltage where the pinacidil-induced current crossed the voltage axis and increased the slope conductance of this current; (4) the pinacidil-induced current was reduced by Ba²⁺ (0.5–1.5 mM) and abolished by intracellular Cs⁺ (125 mM); (5) glibenclamide reversibly reduced or abolished the pinacidil-induced current; (6) the action of pinacidil was decreased by elevating [ATP] in the pipette solution (from 1 to 10 mM); (7) the action of pinacidil was augmented by adding isoproterenol (1 M) to the superfusate or adding cAMP (0.1 mM) to the pipette solution; (8) elevating temperature augmented, and accelerated the onset of, pinacidil's action; (9) pinacidil reversibly decreased the Ca²⁺ -independent transient outward current (I_to1) but augmented the Ca²⁺ -dependent transient outward current (I_to2). Based on these observations, we reached the following conclusions: (1) the main effect of pinacidil is to increase an outward current through the ATP-regulated K⁺ channels; (2) pinacidil's action is modulated by an enzymatic reaction.     

Interferon inhibition of lymphocyte mitogenesis.

Interferon inhibits the early phase of mitogenic action of Concanavalin A on lymphocytes as measured by incorporation of labelled precursors in RNA, DNA or protein. There was no evidence for a role of cyclic AMP in the process of inhibition. Dialysis of interferon against high salt and urea did not separate cell inhibitory activity from antiviral activity. Thus, these results are compatible with the possibility that the antiviral and cell inhibitory activities are in the same molecule. However, further purification of the interferon will be required to verify this assumption.     

Dual regulation of BCR-mediated growth inhibition signaling by CD72

CD72 has been reported to regulate BCR-mediated signals both positively and negatively. SHP-1 and Grb2 bind, respectively, to ITIM1 and ITIM2 of CD72. We generated transformed B cell lines with an immature phenotype following J2 virus infection of splenocytes from CD72(-/-) and wild-type (Wt) mice. The transformed lines were infected with retroviral vectors carrying Tyr (Y) to Phe (F) substitutions in the ITIM sequences (ITIM1 mutated: Y7/F; ITIM2 mutated: Y39/F; and both ITIM mutated: Y7,39/F). Cross-linking of the BCR induced growth inhibition in transfectants expressing Wt CD72, but this response was less sensitive in transfectants with Y7,39/F. The Y7/F transfectants demonstrated the least sensitive response. We were not able to obtain transfectants with Y39/F, suggesting that CD72 associated with SHP-1, but not with Grb2, delivers a strong negative signal. Pre-ligation of CD72, which induces dephosphorylation of the molecule, partially rescued the Wt transfectants from growth inhibition, leading to a growth response profile similar to that of Y7,39/F transfectants. These results suggest that ITIM1/SHP-1 delivers a very strong negative signal that is down-modulated by signals through ITIM2/Grb2, leading to delivery of an attenuated negative signal. Thus, pre-ligation of CD72 results in the manifestation of an ostensible positive signal.     

Role of cAMP phosphodiesterase 4 in regulation of T-cell function

Ligation of both the T-cell receptor (TCR) and the CD28 receptor is required for full T-cell activation to occur. Engagement of the TCR in primary T cells is followed by rapid cAMP production in lipid rafts resulting in raft-associated protein kinase A (PKA) activation and inhibition of proximal T-cell signaling. However, upon TCR and CD28 cross-ligation, beta-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) is recruited to lipid rafts, thus downregulating cAMP levels. Consequently, the activities of both PKA and PDE4 seem to be important for the regulation of TCR-induced signaling and T-cell function. We, therefore, propose a novel role for TCR and CD28 co-stimulation in the downmodulation of TCR-induced cAMP-mediated inhibitory signals through the recruitment of beta-arrestin and PDE4 to lipid rafts, thus allowing a full T-cell response to occur.