Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5.

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.

Preclinical pharmacokinetic analysis of NOV-002, a glutathione disulfide mimetic.

NOV-002 is a glutathione disulfide (GSSG) mimetic that is the subject of clinical investigation in oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Non-linear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of approximately 13 min with an AUC of 1.18 µgh/mL, a C(max) of 2.16 µg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis.

Anion and proton transport in chromaffin granules.

Both intact chromaffin granules and ghosts behave as osmometers in response to the relative internal and external concentrations of solutes. The permeability properties of the granule membrane can therefore be studied by measuring the osmotic consequences of ion permeation. These include lysis of intact granules and shrinking or swelling of ghosts. This approach has been used to identify at least two types of selective anion transport sites in the membrane. One is observed in the presence of K+ ionophores and appears to be electrogenic in nature. It is insensitive to a group of compounds known to inhibit anion transport in erythrocytes. The other site is sensitive to those inhibitors and is involved with electroneutral cotransport with protons. This proton entry can be driven by either the proton-pumping ATPase of the granule membrane or by a simple inward concentration gradient of protons. The proton entry-coupled anion transport site may be structurally related to the ATPase itself based on parallel anion and inhibitor sensitivities of the site and the enzyme. One or both of the anion transport sites described here may play a role in regulating exocytosis from cells based on a chemiosmotic mechanism that has been postulated to be responsible for this process.

Isolation and characterization of a bacteriophage infective for a UDP-galactose-4-epimeraseless mutant of Escherichia coli.

A DNA bacteriophage, designated CP13, was isolated against Escherichia coli J5, a UDP-galactose-4-epimeraseless mutant of E. coli 0111:B4. Bacteriophage CP13 appears to be specific for rough bacterial strains. Adsorption studies with E. coli J5 grown with galactose show that the bacteriophage will not adsorb when complete lipopolysaccharide is present in the cell membrane. This indicates that lipopolysaccharide may be directly or indirectly involved with the receptor site for bacteriophage CP13. The bacteriophage DNA has a G + C content of 52%.

Self-association of synexin in the presence of calcium. Correlation with synexin-induced membrane fusion and examination of the structure of synexin aggregates.

It has been proposed (Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, 2858-2866) that synexin, an adrenal medullary protein that causes Ca2+-dependent aggregation of isolated chromaffin granules, might be the intracellular receptor for Ca2+ in the process of exocytosis. We now report that Ca2+ interacts directly with isolated synexin, inducing rapid self-association. Mg2+ or Sr2+ cannot substitute for Ca2+, and Ba2+ is only weakly effective at stimulating self-association. We have analyzed 90 degree light scattering data to determine a titration curve for the activation of synexin by Ca2+ in the self-association reaction. The curve has a Hill coefficient of 2.3 and is half-maximal at 200 micron Ca2+, which correlates exactly with the Ca2+ titration curve for the aggregation of chromaffin granules by synexin. Electron microscopy of negatively stained samples reveals that synexin monomers associate to form 50 A by 150 A rods which in turn associate side to side and end to end to form bundles of parallel rods. We suggest that synexin exists as a soluble monomer in the cytoplasm of the resting chromaffin cell and that when the cell is stimulated to secrete, the increase in the intracellular concentrations of Ca2+ causes synexin to form rods, similar to those formed in vitro, which bind to chromaffin granule and plasma membranes, joining them together to form the "pentalaminar" fusion complexes characteristic of the first step in exocytosis.

Regulation of release from isolated adrenergic secretory vesicles by ATP-mediated changes in transmembrane potential and anion permeability.

Isolated chromaffin granules release their contents when exposed to calcium, magnesium, ATP, and high levels of chloride ions. The mechanism of release is not well-understood, but changes in anion permeability may be involved. We found that another anion, thiocyanate (SCN-), also activated release in a fashion similar to chloride, while isethionate (HO-CH2-CH2SO3-) an impermeant anion, was inactive. Mg++-ATP was found to activate the uptake of 36Cl and 14C-SCN, leading us to conclude that activation of anion uptake might be involved in the release process. The 36Cl and the 14C-SCN compartments were then compared by studying displacement of the trace anions by excess cold mass. Chloride and SCN displaced large amounts of both 36Cl and 14C-SCN, while isethionate displaced little of either tracer anion. We suggest, on the basis of these data, that ATP-mediated anion uptake may be the basis for the release mechanism. Release may occur as a consequence of anion and subsequent water uptake into granules, resulting in osmotic imbalance and osmotic shock. This may also be of physiologic importance, and we propose a cellular model for secretion based on the biochemical properties of the isolated chromaffin granule.

Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis.

Synexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+-dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis-unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin-induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotic trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or toxicologically active substances for effects on secretory systems.

Tenidap modulates cytoplasmic pH and inhibits anion transport in vitro. II. Inhibition of IL-1 beta production from ATP-treated monocytes and macrophages.

IL-1 beta is an important inflammatory mediator produced by monocytes and macrophages after LPS stimulation. In the absence of a secondary stimulus, however, little IL-1 beta is released into the medium. Previously, ATP was shown to promote the release and proteolytic maturation of IL-1 beta from LPS-stimulated murine peritoneal macrophages. Tenidap, a new anti-inflammatory and antiarthritic agent, inhibited the release and maturation of IL-1 beta induced in vitro by ATP treatment of murine peritoneal macrophages. Tenidap's inhibitory activity was mimicked by other agents that blocked anion transport, such as UK5099 and DIDS. In contrast, cyclooxygenase-inhibiting nonsteroidal anti-inflammatory drugs, such as piroxicam and naproxen, did not impair ATP-induced post-translational processing. Human monocytes responded to LPS to produce IL-1 beta, but externalized little of their newly synthesized cytokine. ATP at concentrations > or = 2 mM promoted IL-1 beta release from these cells. The degree to which the released cytokine was proteolytically processed to its biologically active 17-kDa species, however, depended on the pH of the medium; a greater processing efficiency was observed at slightly acidic (pH 6.9) values. Tenidap and other anion transport inhibitors effectively prevented the ATP response of cultured human monocytes. Likewise, LPS-stimulated human alveolar macrophages responded to ATP by releasing 17-kDa IL-1 beta, and tenidap inhibited this response. The ATP-induced release and maturation of IL-1 beta from human monocytes and macrophages, therefore, was suppressed by anion transport inhibitors, suggesting that anion conductance is a necessary component of the ATP-promoted externalization mechanism. In view of IL-1's importance as an inflammatory mediator, tenidap may demonstrate novel anti-inflammatory activities by virtue of its inhibition of the post-translational release and maturation of this cytokine.

Tenidap modulates cytoplasmic pH and inhibits anion transport in vitro. I. Mechanism and evidence of functional significance.

Tenidap is a novel anti-inflammatory and antiarthritic agent that in clinical studies of rheumatoid arthritis patients, displays symptomatic efficacy superior to nonsteroidal anti-inflammatory drugs (NSAIDs) and equivalent to combinations of NSAIDs and second line agents. Clinical and preclinical biochemical studies have demonstrated that tenidap combines cytokine modulation with suppression of prostaglandin biosynthesis. To better understand tenidap's mechanism of action, in vitro studies of intracellular pH (pHi) were conducted. In cells loaded with the pH-sensitive fluorescence dye 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein, tenidap, but not NSAIDs, caused a rapid and sustained acidification of the cytoplasmic compartment. Tenidap did not act as a proton ionophore, as it did not dissipate the low pH within lysosomes. Mammalian cells regulate pHi through the concerted action of a number of specific transport proteins, including sodium-proton antiporters and chloride-bicarbonate exchangers. Tenidap did not alter pHi via inhibition of the sodium-proton antiporter, but inhibited activity of chloride-bicarbonate exchangers, as did UK5099, a known anion-transport inhibitor that also lowers pHi. This similar activity suggests that the pHi change is coupled to anion transport inhibition. As a result of the pHi change, tenidap affected pH-dependent cellular activities. Tenidap inhibited mannose 6-phosphate receptor-mediated endocytosis, inhibited protein synthesis, and stimulated accumulation of the amino acid leucine. Effects on these cellular processes rapidly reversed when tenidap was removed from the culture medium. Tenidap's in vitro activities were highly dependent on the medium composition; protein content, pH, and bicarbonate concentration all were important factors that influenced activity. These results indicate that tenidap is a potent anion-transport inhibitor and modulator of pHi. Within the appropriate cell or tissue microenvironment, these activities may contribute to tenidap's novel therapeutic profile.

Inhibition of cytokine activation processes in vitro by tenidap, a novel anti-inflammatory agent.

Tenidap is a novel anti-inflammatory and anti-arthritic agent that lowers intracellular pH and suppresses anion transport when applied to cells in vitro. Both of these parameters are known to influence pro-inflammatory cell function. To investigate whether tenidap can modulate cellular responses to cytokine stimulation, several in vitro cytokine-driven assays were characterized with respect to their tenidap sensitivity. Human monocytes treated with granulocyte-macrophage colony stimulating factor (GM-CSF) demonstrated an increased production of IL-6 as well as an increased total translational activity. Tenidap dose-dependently inhibited both cytokine-induced responses; the effect on IL-6, however, occurred at lower tenidap concentrations than those required to prevent the increase in total translational activity. In contrast, the known translational inhibitor cycloheximide did not demonstrate selectivity for IL-6; this agent decreased the GM-CSF-induced increase in total translational activity in parallel with its effects on IL-6. GM-CSF-treated monocytes also produced greater amounts of IL-1 beta in response to LPS stimulation than did non-GM-CSF-treated cells, and tenidap again suppressed this cytokine-induced activation. Human Hep3B cells treated with a combination of interleukin (IL)-1 beta and IL-6 demonstrated an acute phase-type of response. These hepatoma cells increased production of the positive acute phase protein serum amyloid A (SAA) while they decreased production of a negative acute phase protein human serum albumin (HSA). Tenidap dose-dependently inhibited the cytokine-induced increase in SAA production without effecting synthesis of HSA or total TCA-precipitable macromolecules. Importantly, the ability of tenidap to alter these various cytokine responses was not shared with piroxicam, a potent cyclooxygenase inhibitor. Finally, human neutrophils treated with either GM-CSF or tumor necrosis factor (TNF)-alpha demonstrated an increased chloride conductance as measured by the loss of radioactive chloride from 36Cl-loaded cells. When tenidap was included within the medium during cytokine stimulation, loss of radioactive chloride was prevented. Thus, tenidap inhibited the cytokine-induced increase in anion transport. Together, these results indicate that tenidap can suppress cellular activation processes induced by a variety of cytokines. This functional antagonism is not dependent on cyclooxygenase inhibition but, rather, appears to link to tenidap's unique ability to alter ionic homeostasis. These in vitro observations, therefore, may help to explain how this novel anti-inflammatory agent acts to lower acute phase proteins and IL-6 levels in man.

Role of intracellular proteins in the regulation of calcium action and transmitter release during exocytosis.

A central problem in cellular neurobiology is how the process of exocytosis of transmitters and hormones is regulated at the molecular level. Calcium is important in the process and may act by initiating the formation of fusion complexes of secretory granules to each other and to plasma membranes. We have discovered and isolated a new 47,000 MW protein (synexin) from adrenal medulla tissue that fuses chromaffin granule membranes only in the presence of calcium. Synexin activity was detected in a number of secretory tissues including human platelets and bovine brain, and the synexin molecule was found by immunofluorescent cytochemistry to be localized to the cytoplasm of chromaffin cells. Purified synexin molecules were found to self-associate in the presence of Ca++ to form paracrystalline arrays of 50 X 150 A rods, and the association was dependent on [Ca++] in an identical fashion to the Ca++ dependence of granule membrane fusion. On the basis of these data we suggest that synexin may be the intracellular receptor for calcium during exocytosis. However, the actual release event of 'fission' of the secretory vesicle-plasma membrane complex did not appear to be related to synexin action, and we have considered the hypothesis that the chemiosmotic mechanism for ATP, Cl--dependent chromaffin granule lysis might provide the necessary localized force. We have now shown that the granule model successfully predicts the secretory properties of human platelets, bovine parathyroid cells, and bovine chromaffin cells. Like the granule lysis system, secretion from these cells required specific anions in the medium was inhibited by anion transport blocking drugs and proton ionophores, and was suppressed by elevated osmotic strength. We suggest that secretory granules fused to plasma membranes in secreting cells, perhaps by synexin, may experience net solute uptake and subsequently undergo local, outwardly directed osmotic lysis, or exocytosis.

An osmotic mechanism for exocytosis from dissociated chromaffin cells.

Dissociated chromaffin cells from bovine adrenal medulla were stimulated to secrete epinephrine and dopamine beta-hydroxylase with a variety of secretagogues in a study designed to test the hypothesis that the chemiosmotic lysis reaction of isolated chromaffin granules might in some way be related to the mechanism of release during exocytosis. Increasing the osmotic strength of the incubation medium with either NaCl or sucrose led to suppression of secretion of epinephrine from the cells regardless of whether secretion was induced with veratridine or acetylcholine. Suppression of secretion was approximately exponential with respect to osmotic strength. Epinephrine secretion occurred only if the medium contained a permeant anion such as chloride, and secretion induced by veratridine was suppressed when Na isethionate replaced NaCl in the medium. In an extensive study with different monovalent anions veratridine supported epinephrine secretion according to the following activity series: Br-, I-, NO3- greater than methylsulfate, SCN- greater than Cl greater than acetate much greater than isethionate. A similar series, except for the potency of NO3-, was observed with A23187 as agonist. In general, the anion series for granule lysis was analogous. However, there was a poor quantitative correlation between the anion dependence of chemiosmotic granule lysis and the anion dependence of cell secretion. Anion transport inhibitors such as probenecid and pyridoxal phosphate also inhibited secretion while the stilbene disulfonates were inactive. The ineffectiveness of the stilbene disulfonates further distinguished chemiosmotic granule lysis from cell secretion. Secretion of catecholamines, induced by veratridine or nicotine, a cholinergic agonist, was suppressed when NaCl in the medium was replaced by isosmotic sucrose and unexpectedly low levels of dopamine beta-hydroxylase were observed in some cases. In sum, these properties of secreting chromaffin cells resembled some properties of isolated chromaffin granules incubated in ATP and Cl-, but were different in a number of instances. We, therefore, have interpreted our data to indicate that while some mechanistic relationships may indeed exist between the release event in exocytosis from chromaffin cells and the chemiosmotic lysis reaction characteristic of isolated chromaffin granules, an understanding of the energetics of exocytosis awaits the discovery of reasons for the quantitative differences between the two systems.

The in vivo pharmacology of CP-80, 633, a selective inhibitor of phosphodiesterase 4.

The following studies were conducted to characterize the bron-chodilatory and antiinflammatory activity of the novel, selective phosphodiesterase-IV inhibitor, CP-80,633 (2'S)5-[3-(2'-exobicyclo[2.2.1]heptyloxy-4-methoxy-phenyl]te trahydro- 2(1H)-pyrimidone, a compound in clinical development for atopic disease. In IgG1 passively sensitized guinea pigs, aerosolized ovalbumin challenge increases both pulmonary eosinophil peroxidase levels and airway obstruction. CP-80,633, administered before ovalbumin challenge, significantly attenuated both the increase in tissue eosinophil peroxidase levels (ED50 = 1.4 mg/kg, p.o.) and airway obstruction (ED50 = 0.93 +/- 0.14 mg/kg,p.o.) 10 to 30 times more potently than theophyl-line. Intraarterially administered CP-80,633 also reversed an established bronchoconstriction initiated by continuous infusion of histamine to guinea pigs (ED50 of 8.2 micrograms/kg vs. 5.6 mg/kg for theophylline). The antiinflammatory effect of CP-80,633 was also examined in atopic monkeys challenged with Ascaris suum (Ag) aerosol. CP-80,633 (1 mg/kg, qid, s.c., 1 hr before antigen challenge) significantly reduced antigen-induced increases in bronchoalveolar lavage neutrophils (72.8 +/- 15.8% inhibition) and eosinophils (61.1 +/- 5.7% inhibition) 4 hr postchallenge, but did not reduce leukocytes 24 hr postchallenge. CP-80,633 did not inhibit antigen-induced increases in BAL levels of interleukin-1 beta, -6 or -8 as measured by enzyme-linked immunosorbant assay. These results indicate that CP-80,633 possesses bronchodilatory activity in guinea pigs and some antiinflammatory effects in both guinea pigs and monkeys.

Evidence for control of serotonin secretion from human platelets by hydroxyl ion transport and osmotic lysis.

Serotonin secretion from human platelets, stimulated either by thrombin or the calcium ionophore A23187, was found to be inhibited by anion transport blocking drugs such as 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), pyridoxal phosphate, probenecid, and suramin. These drugs have previously been shown to inhibit ATP-evoked release of epinephrine from isolated chromaffin granules by blocking chloride uptake and subsequent osmotic lysis. However, in contrast to granule release, platelet secretion was insensitive to chloride and, instead, was dependent on OH-. Platelet release was suppressed by low pH, and inhibition by the transport blocking drugs was competitive only with respect to OH-. Serotonin release from platelets was also suppressed by increasing extracellular osmotic strength, and the relationship between suppression and external osmotic strength was quantitatively similar to that observed in the case of chromaffin granules. We conclude that platelet exocytosis could occur when serotonergic granules are closely juxtaposed to the plasma membrane, thus exposing the granule anion transport site to the more alkaline medium. Secretion of serotonin could occur as a consequence of OH- transport and osmotic lysis of the granule-plasma membrane complex, analogous to the chemiosmotic mechanism of chloride-dependent epinephrine release from isolated chromaffin granules.

Role of anions in parathyroid hormone release from dispersed bovine parathyroid cells.

It is known that permeant anions are required for the release of epinephrine from isolated chromaffin granules and of serotonin from intact platelets. We have now investigated the role of anions in the release of a polypeptide hormone, parathyroid hormone, from dispersed bovine parathyroid cells. The release is inhibited 60%-80% by decreasing either [Cl-] or [OH-] and 60%-70% by replacement of NaCl with the impermeant anion isethionate. By contrast, substitution of various monovalent cations in the medium had no effect on the release. Disodium 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and probenecid, which are known to block anion transport in the erythrocyte, also cause a dose-dependent 90%-100% inhibition of release. Moreover, kinetic analysis of inhibition by probenecid suggests that it is competitive with respect to either OH- or Cl-. These results suggest that anions and the anion transport system may play a role in exocytosis of a polypeptide hormone. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone was was also found to block hormone release, and the possibility is discussed of a "chemosmotic" mechanism for exocytosis in this system similar to that previously postulated for chromaffin granules and platelets.

A role for bombesin in sensory processing in the spinal cord.

Bombesin (BN)-containing neuronal processes were demonstrated in laminae I and II of the dorsal horn of the cat, rat, and mouse spinal cord by immunocytochemistry and radioimmunoassay. Dorsal rhizotomy in the cat resulted in a marked decrease in BN immunoreactivity in the dorsal horn indicating that BN is contained in primary sensory afferents. BN-binding sites were also localized in superficial laminae of the dorsal horn. The presence of both BN and BN-binding sites in the dorsal horn suggested that BN may be involved in sensory processing in the spinal cord. Consistent with this hypothesis, it was demonstrated that an injection of BN into the spinal cord caused a biting and scratching response indicative of sensory stimulation. The effect was similar to that observed after injection of substance P into the cord with the exception that the BN effect lasted about 100 times longer than that induced by substance P. Taken together, these data indicate that BN may be a neurotransmitter of primary sensory afferents to the spinal cord.

In vitro pharmacology of the novel phosphodiesterase type 4 inhibitor, CP-80633.

We present the in vitro pharmacology of a novel adenosine 3'-5' -cyclic monophosphate-specific phosphodiesterase (PDE) type 4 inhibitor, CP-80633 ((2'S)5-[3-(2' -exobicyclo[2.2.1]-heptyloxy)4-methoxyphenyl] tetrahydro-2(1H)-primidone), which has shown efficacy in phase II clinical trials for atopic dermatitis. CP-80633 inhibits PDE4 isozymes (human lung IC50 = 1.27 microM) in the absence of effects on PDE1, PDE2, PDE3 and PDE5 isozymes (IC50 > 100 microM). It exhibits no significant selectivity for any single cloned PDE4A, B, C or D isoform. CP-80633 inhibits adenosine 3'-5'-cyclic monophosphate hydrolysis in partially purified human peripheral blood monocyte cytosol (IC50 = 3.52 microM), eosinophil membrane (IC50 = 1.10 microM) and T cell membrane (IC50 = 2.28 microM) preparations. Inhibition of eosinophil PDE4 adenosine 3'-5'-cyclic mono-phosphate hydrolysis by CP-80,633 occurs in a noncompetitive manner. Unlike theophylline, CP-80,633 is inactive against ratrain adenosine (A1,A2) receptors. Consistent with its action as a PDE4 inhibitor in whole cells, CP-80633 potentiates PGE1 dependent increases in adenosine 3'-5'-cyclic monophosphate levels in human U937 cells, and in human eosinophils, monocytes and T cells (EC200 approximately 1.0 microM). Consequently, CP-80633 inhibits many inflammatory cell functions including 1) human eosinophil superoxide anion production (IC50 < 0.6 microM), 2 C5a-(IC50 = 0.40 microM) and LTB4-(IC50 = 0.20 microM) mediated guinea pig peritoneal eosinophil chemotaxis and 3) lipopolysac-charide-induced tumor necrosis factor-alpha release from human monocytes (IC50 = 0.219 microM). These data clearly demonstrate that CP-80633 is a selective inhibitor of PDE4 isozymes, and support its potential use as a therapeutic agent for a number of inflammatory and immune disorders.

The phosphodiesterase type 4 (PDE4) inhibitor CP-80,633 elevates plasma cyclic AMP levels and decreases tumor necrosis factor-alpha (TNFalpha) production in mice: effect of adrenalectomy.

Rolipram was previously reported to elevate plasma cyclic adenosine 3',5'-monophosphate (cAMP) and inhibit serum tumor necrosis factor-alpha (TNF-alpha) production in mice. CP-80,633, a new cyclic nucleotide phosphodiesterase (PDE4) inhibitor, has been shown to augment intracellular cAMP levels and to inhibit TNFalpha release from human monocytes in vitro. This study was undertaken to determine the effect of p.o. CP-80,633 on plasma cAMP levels and lipopolysaccharide-induced TNFalpha production in mice with and without adrenal glands. CP-80,633 dose-dependently (3-32 mg/kg p.o.) elevated plasma cAMP levels and decreased systemic TNFalpha production in response to i.p. injection of lipopolysaccharide. Elevated plasma cAMP levels can be detected for up to 4 hr. CP-80,633 (10 mg/kg p.o.) caused a 6-fold increase in the plasma cAMP level, a 2-fold increase in the plasma epinephrine level and a greater than 95% reduction in TNFalpha production. Unlike CP-80,633, neither vinpocetine, dipyridamole, SKB-94,120 nor zaprinast, at 100 mg/kg p.o., modified the cAMP response, which suggests that this response is mediated by inhibition of PDE4. Adrenalectomy reduced the cAMP response and completely blocked the epinephrine response; however, the levels of plasma cAMP in the CP-80,633-treated mice (10 mg/kg p.o.) remained elevated (vehicle: 47.3 +/- 6.8 vs. CP-80,633: 98.4 +/- 10.3 pmol/ml, n = 7, P < .05). This effect is mimicked by treatment of control mice with propranolol, which demonstrates that beta adrenoreceptors contribute to the cAMP response. Removal of adrenal glands significantly increased the LPS-induced elevation of serum TNFalpha. The ability of CP-80,633 to block the TNFalpha response was only slightly affected by adrenalectomy (ED50 = 1.2 mg/kg in controls vs. 3.9 mg/kg in adrenalectomized mice). Taken together, these results show that CP-80,633, when given p.o. to mice, is capable of elevating plasma cAMP and inhibiting TNFalpha production and that adrenal catecholamines contribute significantly to the effect of CP-80,633 on the cAMP response but only slightly to its effect on the systemic TNFalpha response.