Cadmium-induced calcium release and prostaglandin E2 production in neonatal mouse calvaria are dependent on cox-2 induction and protein kinase C activation

The mechanisms by which cadmium (Cd) causes skeletal impairment have not been fully clarified. Release of calcium from neonatal mouse calvaria in organ culture is stimulated by submicromolar concentrations of Cd, an effect that is associated with increased production of prostaglandin E₂ (PGE₂). The prostaglandin-synthesising enzyme cyclooxygenase (cox) exists in two forms, one constitutive (cox-1) and the other inducible (cox-2). Cox-2 can be induced by mitogenic stimuli and inflammatory cytokines, such as parathyroid hormone (PTH), interleukin-1α and tumour necrosis factor-α. Cd potently activates protein kinase C (PKC), which in turn induces cox-2 production in several cell types. Our aim was to determine whether Cd-induced Ca release and PGE₂ production in neonatal mouse calvaria involve induction of cox-2 and, if so, to ascertain whether that effect is mediated by activation of PKC. Cd dose-dependently stimulated Ca release from cultured neonatal mouse calvaria, with a maximal effect at 0.4–0.8 μM. Different sensitivity was observed to Cd-induced Ca release between two breeds of mice suggesting that the susceptibility to Cd may be genetically determined. Dexamethasone (10 μM) added to the culture medium abolished the Ca releasing effect of Cd, an effect not overcome by addition of arachidonic acid (10 μM). The cox-2-selective inhibitors NS-398 and DFU and the less selective inhibitor meloxicam, potently impeded Cd-induced Ca release (IC₅₀ of 1 nM, 41 nM and 7 nM, respectively) and calvarial production of PGE₂. Cd-induced and phorbol 12-myristate 13-acetate (PMA; 20 nM)-induced Ca release was inhibited by the PKC inhibitor calphostin C (0.5 μM) and by NS-398. The effects of PMA and Cd on Ca release were not additive, suggesting that both operated via the PKC pathway. We suggest that Cd-induced Ca release from neonatal mouse calvaria in culture depends on induction of cox-2 that occurs via the PKC signalling pathway. Received: 10 February 1999 / Accepted: 23 March 1999     

Stimulation of Prostaglandin Production by Quinolone Phototoxicity in Balb/c 3T3 Mouse Fibroblast Cells in Vitro

Sparfloxacin (SPFX) and levofloxacin (LVFX) with ultraviolet-A(UVA) irradiation have been reported to induce skin inflammationdue to phototoxicity in Balb/c mice. We examined the productionof arachidonic acid metabolites induced by quinolone phototoxicityin Balb/c 3T3 mouse fibroblast cells in vitro. The cells weresimultaneously treated with SPFX or LVFX at 1,10, or 100 µMand UVA irradiation for 5 min (0.5 J/cm²). They were then culturedin quinolone-free medium for 24 hr, and the concentrations ofprostaglandin E₂ (PGE₂ 6-ketoprostaglandin F₁ (6-keto-PGF₁),and leukotriene B₄ (LTB₄) in the incubation medium were measured.Furthermore, the effect of quinolone photoproducts on the productionof the inflammatory mediators and that of indomethacin on PGE₂level were also examined. Treatment with SPFX at 100 µMplus UVA irradiation markedly increased levels of PGE₂ and 6-keto-PGF₁but not that of LTB SPFX or LVFX alone at up to 100 µM,100 µM SPFX, or 100µM LVFX, or less plus UVA irradiation,or UVA-preirradiated quinolone up to 100µM had no effect.indomethacin even at 0.1 µM completely inhibited the PGE₂elevation induced by 100 µM SPFX with UVA. These resultssuggest that PGs released from dermal fibroblasts in the simultaneouspresence of quinolone and UVA could contribute in part to thedevelopment of skin inflammation in vivo.     

Effects of propofol on the leukocyte nitric oxide pathway: in vitro and ex vivo studies in surgical patients

This study was designed to evaluate the mechanism by which propofol modifies leukocyte production of nitric oxide (NO) in humans. In vitro experiments used whole blood from healthy volunteers (n = 10 samples/experiment). Ex vivo experiments studied the effects of an intravenous dose of 2.5 mg propofol per kilogram body weight followed by intravenous infusion of 4 mg kg⁻¹ h⁻¹ in surgical patients in ASA class I or II (n = 20). In whole blood, neutrophils and plasma, we measured NO production and the activities of the enzymes nitric oxide synthase [inducible (iNOS) and constitutive (cNOS)] and cyclooxygenase [constitutive (COX-1) and inducible (COX-2)]. Concentrations of interleukins (IL-1β, IL−6, and IL−10) and tumor necrosis factor-alpha (TNFα) were measured in plasma. In blood from healthy donors, propofol increased NO production and cNOS activity. The concentration of propofol that increased NO production by 50% (EC₅₀) was 23.5 μM, and the EC₅₀ of propofol for cNOS was 18.6 μM. In blood from surgical patients, propofol increased NO production by 52% and cNOS activity by 57%. Propofol inhibited iNOS activity in vitro; the concentration that reduced activity by 50% (IC₅₀) was 19.9 μM. In surgical patients propofol inhibited iNOS activity by 53%. COX-1 and COX-2 activities were inhibited in vitro (IC₅₀ 32.6 and 187 μM, respectively) and in surgical patients (53 and 81% inhibition, respectively). Plasma concentrations of IL-1β, IL-6, and TNFα were significantly reduced in surgical patients (32, 23, and 21% inhibition, respectively). None of these parameters were modified in a group of patients (n = 10) anesthetized with sevoflurane. We conclude that propofol stimulated constitutive NO production and inhibited inducible NO production, possibly by curtailing the stimulation of iNOS by inflammatory mediators in surgical patients.     

Positive allosteric modulatory effects of ajulemic acid at strychnine-sensitive glycine α<Subscript>1</Subscript>- and α<Subscript>1</Subscript>β-receptors

The synthetic cannabinoid ajulemic acid (CT-3) is a potent cannabinoid receptor agonist which was found to reduce pain scores in neuropathic pain patients in the absence of cannabis-like psychotropic adverse effects. The reduced psychotropic activity of ajulemic acid has been attributed to a greater contribution of peripheral CB receptors to its mechanism of action as well as to non-CB receptor mechanisms. Loss of inhibitory synaptic transmission within the dorsal horn of the spinal cord plays a key role in the development of chronic pain following inflammation or nerve injury. Inhibitory postsynaptic transmission in the adult spinal cord involves mainly glycine. As we hypothesised that additional non-CB receptor mechanisms of ajulemic acid might contribute to its effect in neuropathic pain, we investigated the interaction of ajulemic acid with strychnine-sensitive α₁- and α₁β-glycine receptors by using the whole-cell patch clamp technique. Ajulemic acid showed a positive allosteric modulating effect in a concentration range which can be considered close to clinically relevant concentrations (EC₅₀ values: α₁ = 9.7 ± 2.6 μM and α₁β = 12.4 ± 3.4 μM). Direct activation of glycine receptors was observed at higher concentrations above 100 μM (EC₅₀ values: α₁ = 140.9 ± 21.5 μM and α₁β = 154.3 ± 32.1 μM). These in vitro results demonstrate that ajulemic acid modulates strychnine-sensitive glycine receptors in clinically relevant concentrations.     

Endotoxin induces the expression of prostaglandin H synthase-2 and eicosanoid formation in cells of the human monocytic cell line Mono Mac 6

In controlling inflammation, monocytes and macrophages not only secrete various cytokines but also eicosanoids. Since the human monocytic cell line, Mono Mac 6, represents cells at the stage of mature blood monocytes, cells of this line were investigated to find out whether they also resemble mature monocytes with respect to inflammation. In response to lipopolysaccharide (LPS), Mono Mac 6 cells secreted large amounts of eicosanoids, 6-keto-prostaglandin F_1α (6k-PGF_1α), thromboxane B₂ (TXB₂) and prostaglandin E₂ (PGE₂). The considerable increase in eicosanoid secretion is attributed to the expression of the inducible isoform of the prostaglandin H synthase (PGHS-2) since the PGHS-2-specific steady-state mRNA level, PGHS-2 protein, and the enzyme activity of PGHS were strikingly increased in the absence of detectable PGHS-1 protein. PGHS-2 expression was inhibited by either dexamethasone or cycloheximide. The non-steroidal anti-inflammatory drug (NSAID) diclofenac inhibited the increased PGHS activity. The expression of the PGHS-2 gene was accompanied by the formation of TNFα, IL-6 and IL-8, but not by IL-1β.     

Interleukin-1β and tumor necrosis factor-α inhibit the release of [3H]-Noradrenaline from isolated human atrial appendages

In the present study, we have investigated the ability of human recombinant interleukin-1β (hIL-1β) and human recombinant tumor necrosis factor-α (hTNF-α) to modulate the stimulation-induced (S-I) outflow of [³H]-noradrenaline ([³H]-NA) from isolated superfused human atria. Pieces of human right atrial appendages were excised during routine cardiac surgery. Tissues were incubated with [³H]-NA (0.2 μmol/l) for 30 min at 37°C, then inserted in a Brandel suprafusion system where the radioactivity was washed for 75 min with a Krebs-Henseleit solution at a rate of 0.4 ml/min. Thereafter, the effluent was collected for the remainder of the protocol during which two trains of electrical stimulation (50 mA intensity, 5 Hz frequency, 60 s duration, 2 ms pulses) were delivered at 10 min and 45 min (short protocol) or 85 min (long protocol). The effect of drugs on the S-I outflow of [³H]-NA was determined by adding drugs 20 min (short protocol) or 60 min (long protocol) before the second stimulation. Experiments were carried out in the continuous presence of desipramine (1 μmol/l) to prevent neuronal NA reuptake. The results showed that in human atrium, hIL-1β (3 ng/ml) and hTNF-α (0.5 ng/ml) significantly inhibited the S-I release of [³H]-NA. The inhibitory effect of hIL-1β was blocked by human recombinant IL-1 receptor antagonist (50 ng/ml), and by the cyclooxygenase inhibitor, diclofenac (1 μmol/l), suggesting that hIL-1β inhibited NA release through the formation of prostaglandins. The ability of hIL-1β and hTNF-α to inhibit NA release suggest that mediators of the immune system produced locally may modulate the activity of the sympathetic nervous system in human atrial appendages. Received: 11 October 1996 / Accepted: 27 November 1996     

α2-Adrenoceptors in opossum kidney cells couple to stimulation of mitogen-activated protein kinase independently of adenylyl cyclase inhibition

We have compared the effects of adrenaline on activation of mitogen-activated protein kinase (MAP kinase), cyclic AMP accumulation and [³H]thymidine uptake in OK cells, a cell line derived from proximal tubules of the opossum kidney. Effects of serotonin and the direct protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), were also studied. Adrenaline transiently (peak at 5min, return to baseline by 30min) and concentration-dependently (EC₅₀ between 10 and 100nM) stimulated MAP kinase activity. Maximal stimulation was approximately 100% above basal and was similar to the effects of 1μM serotonin or 1μM PMA. MAP kinase activation by adrenaline was inhibited by 10μM phentolamine or 1μM yohimbine but not significantly affected by 100nM prazosin or 200nM pindolol. The selective α₂-adrenoceptor agonist UK 14,304 (10μM) also stimulated MAP kinase activity. Activation of the 42 and 44kDa ERK forms of MAP kinase was demonstrated by immunoblot analysis. The effect of adrenaline and UK 14,304 on MAP kinase was inhibited by pertussis toxin pretreatment and by the MAP kinase kinase inhibitor, PD 98059 (100μM). Stimulation of MAP kinase activity was independent of cellular cAMP levels and was not affected by protein kinase C down-regulation. Adrenaline, UK 14,304, serotonin, and PMA stimulated [³H]thymidine uptake, an effect inhibited by PD 98059. We conclude that adrenaline stimulates MAP kinase activity in OK-cells via α₂-adrenoceptors and pertussis sensitive G proteins. While this occurs independently of cellular cAMP levels and protein kinase C, it involves the MEK1 form of MAP kinase kinase and the ERK forms of MAP kinase. This activation results in enhanced cellular proliferation as assessed by [³H]thymidine uptake. Received: 2 April 1997 / Accepted: 29 April 1997     

Phospholipase A2 inhibitors p-bromophenacyl bromide and arachidonyl trifluoromethyl ketone suppressed interleukin-2 (IL-2) expression in murine primary splenocytes

Phospholipase A₂ (PLA₂) has been postulated to play a role in the regulation of cytokine expression. Therefore, the objective of the present study was to investigate the effects of PLA₂ inhibitors p-bromophenacyl bromide (BPB) and arachidonyl trifluoromethyl ketone (AACOCF₃) on interleukin-2 (IL-2) expression in murine primary splenocytes. Pretreatment of the splenocytes with both BPB and AACOCF₃ suppressed phorbol 12-myristate 13-acetate plus ionomycin-induced IL-2 secretion in a concentration-dependent manner. Inhibition >90% of IL-2 secretion was observed at 1 μM BPB and 10 μM AACOCF₃ compared to the respective vehicle control. Likewise, IL-2 steady-state mRNA expression was inhibited by both PLA₂ inhibitors in a concentration-dependent fashion with >90% inhibition at 1 μM BPB and 20 μM AACOCF_3. Taken together, these data demonstrated that PLA₂ inhibitors BPB and AACOCF₃ are robust inhibitors of IL-2 expression at both the mRNA and protein levels in murine splenocytes. Moreover, these findings suggest that drugs and chemicals which inhibit PLA₂ may have marked effects on T-cell function. Received: 27 July 1998 / Accepted: 24 November 1998     

Dexmedetomidine inhibits muscarinic type 3 receptors expressed in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes and muscarine-induced intracellular Ca<Superscript>2+</Superscript> elevation in cultured rat dorsal root ganglia cells

Dexmedetomidine, an α₂-adrenoceptor agonist, has been approved for clinical use, although the mechanism of dexmedetomidine action has not been fully elucidated. Several studies have shown that G protein-coupled receptors (GPCRs) are recognized as targets for anesthetics and analgesics. Therefore, it is of interest to determine whether dexmedetomidine affects the function of GPCRs other than the α₂-adrenoceptor. We examined the effects of dexmedetomidine on M₁, M₃, 5-HT_2C, substance P, and orexin 1 receptors in Xenopus oocytes expressing individual receptors. In addition, we investigated the effects of dexmedetomidine on muscarinic receptor-mediated changes in [Ca²⁺]_i in the dorsal root ganglia (DRG) of 3-week-old Wister rats. Dexmedetomidine did not affect the 5-HT_2C-, or substance P-induced Cl⁻ currents and had little inhibition on the orexin A-induced current in oocytes expressing the respective receptors. The compound also had little effect on the acetylcholine (ACh, 1 μM)-induced Ca²⁺-activated Cl⁻ currents in Xenopus oocytes expressing M₁ receptors. In contrast, dexmedetomidine inhibited the ACh-induced currents in Xenopus oocytes expressing M₃ receptors; 1 nM, 10 nM, 100 nM, and 1 μM dexmedetomidine reduced the current to 66.5 ± 4.8, 51.3 ± 12, 34.6 ± 11, and 26.8 ± 6.4% of the control value, respectively (EC₅₀ = 3.5 ± 0.7 nM). Dexmedetomidine reduced the ACh-induced Cl⁻ currents after treatment with the selective protein kinase C inhibitor GF109203X. Moreover, the compound inhibited the muscarinic receptor-mediated increases in [Ca²⁺]_i in cultured DRG cells in a concentration-dependent manner. Dexmedetomidine inhibits the function of M₃ receptors, in addition to its agonistic effects on α₂-adrenoceptors, which provides further insight into the pharmacological properties of dexmedetomidine.     

Potent mechanism-based inhibition of human CYP3A in vitro by amprenavir and ritonavir: comparison with ketoconazole

Objective: Biotransformation of triazolam to its α-hydroxy and 4-hydroxy metabolites by human liver microsomes in vitro was used as an index of human cytochrome P ₄₅₀ 3A (CYP3A) activity. Results: The reaction was strongly inhibited by co-incubation with the viral protease inhibitors ritonavir (IC₅₀=0.14 μM) and amprenavir (IC₅₀=2.5–2.9 μM), and by the azole derivative ketoconazole (IC₅₀ = 0.07 μM). Pre-incubation of microsomes with ritonavir or amprenavir increased inhibitory potency (IC₅₀ reduced to 0.07 μM and 1.4 μM, respectively). This was not the case with ketoconazole. Conclusions: Thus, ritonavir and amprenavir are highly potent mechanism-based inhibitors of human CYP3A isoforms. Received: 11 January 2000 / Accepted in revised form: 9 March 2000     

The effects of celecoxib, a COX-2 selective inhibitor, on acute inflammation induced in irradiated rats

The potential value of selective and non-selective COX-2 inhibitors in preventing some of the biochemical changes induced by ionizing radiation was studied in rats exposed to carrageenan-induced paw edema and 6-day-old air pouch models. The animals were exposed to different exposure levels of γ-radiation, namely either to single doses of 2 and 7.5 Gy or a fractionated dose level of 7.5 Gy delivered as 0.5 Gy twice weekly for 7.5 weeks. The inflammatory response produced by carrageenan in irradiated rats was markedly higher than that induced in non-irradiated animals, and depended on the extent of irradiation. Celecoxib, a selective COX-2 inhibitor, in doses of 3, 5, 10, and 15 mg/kg was effective in reducing paw edema in irradiated and non-irradiated rats in a dose-dependent manner as well as diclofenac (3 mg/kg), a non-selective COX inhibitor. Irradiation of animals before the induction of the air pouch by an acute dose of 2 Gy led to a significant increase in leukocytic count, as well as in the level of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), LTB₄, PGE₂ (as an index of COX-2 activity), TXB₂ (as an index of COX-1 activity), and the plasma level of MDA. This increase in level of these parameters was more marked than that observed in the non-irradiated animals subjected to the inflammagen. The blood GSH level was not affected by the dose of irradiation used, whereas superoxide dismutase (SOD) activity was suppressed. In many respects, celecoxib (5 mg/kg) was as potent as diclofenac in decreasing the elevated levels of IL-6, IL-1β, TNF-α, LTB₄, PGE₂, but lacked any significant effect on TXB₂ level. Since it is mostly selective for COX-2 with a rare effect on COX-1 enzyme, both drugs at the selected dose levels showed no effect on level of MDA, GSH, and SOD activity.     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

Modulation of immunoreactive protein kinase C-α and β isoforms and G proteins by acute and chronic treatments with morphine and other opiate drugs in rat brain

The abundance of protein kinase C-α and β isoforms (PKC-αβ), PKC-α messenger (m) RNA and guanine nucleotide-binding G protein subunits (Gα_i1/2, Gα_o and Gβ) were quantitated in the rat cerebral cortex after acute and chronic treatments with various opiate drugs. Acute (100mg/kg for 2h) and chronic (10 to 100mg/kg for 5 days) treatment with morphine decreased similarly the immunoreactivity of PKC-αβ (28% and 32%, respectively). Acute (2h) and chronic treatment (5 days) with other μ-agonists heroin (30mg/kg and 10 to 30mg/kg) and methadone (30mg/kg and 5 to 30mg/kg) also induced similar decreases of PKC-αβ (acute: 25% and 23%; chronic: 28% and 18%). After the chronic treatments, spontaneous (48h) or naloxone (2mg/kg)-precipitated opiate withdrawal (2h) resulted in up-regulation of PKC-αβ above control levels (30-38%), and in the case of morphine withdrawal in a concomitant marked increase in the expression of PKC-α mRNA levels (2.3-fold). Acute (2h) treatments with pentazocine (80mg/kg, mixed κ/δ-agonist and μ-antagonist), spiradoline (30mg/kg, selective κ-agonist) and [D-Pen², D-Pen⁵] enkephalin (14nmol i.c.v., selective δ-agonist) induced significant decreases of PKC-αβ (19–33%). Chronic (5 days) treatment with pentazocine (10 to 80mg/kg), but not spiradoline (2 to 30mg/kg), also induced a similar decrease of PKC-αβ (35%). In pentazocine- or spiradoline-dependent rats, naloxone (2mg/kg) did not induce up-regulation of brain PKC-αβ. Acute (10mg/kg for 2h) and chronic (2×10mg/kg for 5 and 14 days) treatment with naloxone did not alter PKC-αβ immunoreactivity. Chronic, but not acute, treatment with μ-agonists (morphine, heroin and methadone) increased the immunoreactivities of Gα_i1/2 (33-37%), Gα_o (25-41%) and Gβ (10-33%) protein subunits. In heroin- and methadone-dependent rats naloxone (2mg/kg)-precipitated withdrawal (2h) did not modify the up-regulation of these G proteins induced by chronic μ-opiate treatment. In marked contrast to μ-agonists, chronic treatment with high doses of pentazocine and spiradoline or acute treatment with [D-Pen², D-Pen⁵] enkephalin did not result in up-regulation of these G protein subunits. After chronic treatment with μ-agonists, significant negative correlations were found when the percentage changes in immunoreactivity of PKC-αβ were related to the percentage changes in immunoreactivity of Gα_i1/2 (r =-0.53, n = 29) and Gβ (r =-0.41, n = 24) in the same brains. PKC-αβ abundance did not correlate significantly with the density of Gα_o (r =-0.21, n = 28). Together the results indicate that the brain PKC-αβ system may play a major regulatory role in opiate tolerance and dependence. Moreover, the possible in vivo cross-communication between this regulatory enzyme and specific inhibitory G proteins may also be of relevance in the cellular and molecular processes of opiate addiction. Received: 15 July 1996 / Accepted: 22 November 1996     

Modulation of tumor necrosis factor α expression in mouse brain after exposure to aluminum in drinking water

Aluminum, a known neurotoxic substance and a ground-water pollutant, is a possible contributing factor in various nervous disorders including Alzheimer's disease. It has been hypothesized that cytokines are involved in aluminum neurotoxicity. We investigated the alterations in mRNA expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interferon γ (IFNγ), cytokines related to neuronal damage, in cerebrum and peripheral immune cells of mice after exposure to aluminum through drinking water. Groups of male BALB/c mice were administered aluminum ammonium sulfate in drinking water ad libitum at 0, 5, 25, and 125 ppm aluminum for 1 month. An additional group received 250 ppm ammonium as ammonium sulfate. After treatment, the cerebrum, splenic macrophages and lymphocytes were collected. The expression of TNFα mRNA in cerebrum was significantly increased among aluminum-treated groups compared with the control, in a dose-dependent manner. Other cytokines did not show any aluminum-related effects. In peripheral cells, there were no significant differences of cytokine mRNA expressions among treatment groups. Increased expression of TNFα mRNA by aluminum in cerebrum may reflect activation of microglia, a major source of TNFα in this brain region. Because the aluminum- induced alteration in cytokine message occurred at aluminum concentrations similar to those noted in contaminated water, these results may be relevant in considering the risk of aluminum neurotoxicity in drinking water. Received: 27 May 1999 / Accepted: 20 July 1999     

Blockade of protein kinase C is involved in the inhibition by cycloheterophyllin of neutrophil superoxide anion generation

Cycloheterophyllin, a prenylflavone, inhibited the superoxide anion (O₂ ^-)generation from formylmethionyl-leucyl-phenylalanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils in a concentration-dependent manner with IC₅₀ values of 47.0 ± 5.0 and 1.7 ± 0.4 μM, respectively. Cycloheterophyllin had no effect on O₂ ^.- generation in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. Cycloheterophyllin exerted a concentration-dependent inhibition of neutrophil cytosolic protein kinase C (PKC) and rat brain PKC, but had no effect on porcine heart protein kinase A (PKA). Unlike staurosporine, cycloheterophyllin did not affect the trypsin-treated rat brain PKC. [³H]Phorbol 12,13-dibutyrate ([³H]PDB) binding to neutrophil cytosolic PKC was significantly suppressed by cycloheterophyllin. However, cycloheterophyllin had negligible effect on the PMA-induced membrane translocation of PKC-β and PKC-δ in neutrophils. Moreover, the fMLP-induced [Ca²⁺]_i elevation and inositol trisphosphate (IP₃) formation of neutrophils were not affected by cycloheterophyllin at concentrations which significantly suppressed the O₂ ^.- generation. In cell-free system, addition of arachidonate (AA) into the mixture of cytosol and membrane fractions of the resting neutrophils to make NADPH oxidase assembly and activation. Cycloheterophyllin had no effect on O₂ ^.- generation in AA-activated cell-free system. These results suggest that the suppression of PKC activity through the interaction with the regulatory region of PKC is involved in the inhibition by cycloheterophyllin of the O₂ ^.- generation in rat neutrophils. Received: 28 October 1996 / Accepted: 7 February 1997     

Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics

Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E₁- (PGE₁), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate (4αPDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE₁, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism. Received: 20 May 1996/Final version: 6 November 1996     

Inhibitory H3 receptors on sympathetic nerves of the pithed rat: activation by endogenous histamine and operation in spontaneously hypertensive rats

Our previous results demonstrate the occurrence of presynaptic inhibitory histamine H₃ receptors on sympathetic neurons innervating resistance vessels of the pithed rat. The present study, in which new H₃ receptor ligands with increased potency and selectivity (imetit, clobenpropit) were used, was designed to further explore the role of H₃ receptors in the regulation of the rat cardiovascular system. In particular we were interested whether these receptors may be activated by endogenous histamine and whether they are detectable in an experimental model of hypertension. All experiments were performed on pithed and vagotomized rats treated with rauwolscine 1 μmol/kg. In normotensive Wistar rats the electrical (1 Hz, 1 ms, 50 V for 20 s) stimulation of the preganglionic sympathetic nerve fibres increased diastolic blood pressure by about 35 mmHg. Two H₃ receptor agonists, R-(–)-α-methylhistamine and imetit, inhibited the electrically induced increase in diastolic blood pressure in a dose-dependent manner. The maximal effect (about 25%) was obtained for R-(–)-α-methylhistamine at about 10 μmol/kg and for imetit at about 1 μmol/kg. Two H₃ receptor antagonists, thioperamide 1 μmol/kg and clobenpropit 0.1 μmol/kg, attenuated the inhibitory effect of imetit. The neurogenic vasopressor response was increased by about 15% by thioperamide 1 μmol/kg and clobenpropit 0.1 μmol/kg and decreased by 25% by the histamine methyltransferase inhibitor metoprine 37 μmol/kg. R-(-)-α-Methylhistamine, imetit, thioperamide, clobenpropit and metoprine did not affect the vasopressor response to exogenously added noradrenaline 0.01 μmol/kg (which increased diastolic blood pressure by about 40 mmHg). Metoprine had only a very low affinity for H₃ binding sites (labelled by ³H-N^α-methylhistamine; pK_i 4.46). In pithed Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats, electrical (1 Hz, 1 ms, 50 V for 10 s) stimulation increased diastolic blood pressure by 28 and 37 mmHg, respectively. Imetit inhibited the neurogenic vasopressor response to about the same extent in WKY and SHR rats (maximal effect of about 30%). The inhibitory influence of imetit was diminished by thioperamide 1 μmol/kg to about the same degree in rats of either strain. The present study confirms the occurrence of presynaptic H₃ receptors on sympathetic nerve fibres involved in the inhibition of the neurogenic vasopressor response. Moreover, it demonstrates that these H₃ receptors are probably activated by endogenous histamine and appear to be operative in hypertension. Received: 25 July 1996 / Accepted: 24 October 1996     

Prejunctional histamine H3-receptors inhibit electrically evoked endogenous noradrenaline overflow in the portal vein of freely moving rats

The effects of intra-arterial injection of different doses of the selective histamine H₃-receptor agonist R-α-methylhistamine and the selective histamine H₃-receptor antagonist thioperamide on basal and electrically evoked noradrenaline overflow in the portal vein as well as on mean arterial pressure (MAP) and heart rate (HR) were investigated in permanently instrumented freely moving rats. R-α-Methylhistamine (0.01, 0.1 and 1 μmol/kg) inhibited the evoked noradrenaline overflow up to 43%, the ED₅₀ value being 0.013 μmol/kg. Thioperamide (0.1, 0.5 and 1.0 μmol/kg) antagonized the effect of 1.0 μmol/kg R-α-methylhistamine dose-dependently, evoked overflow returning to control values at 1.0 μmol/kg of the antagonist; thioperamide alone had no effect on electrically evoked noradrenaline overflow. Basal noradrenaline levels, blood pressure and heart rate were not at all influenced by R-α-methylhistamine and thioperamide, alone or in combination. The results clearly show the presence of prejunctional histamine H₃-receptors inhibiting the electrically evoked noradrenaline overflow from vascular sympathetic nerve terminals in the portal vein of freely moving rats. Received: 27 August 1996 / Accepted: 14 October 1996