A progesterone-induced protein increases the synthesis of asymmetric antibodies.

The immunological effects of progesterone are mediated by a 34-kDa protein named the progesterone-induced blocking factor (PIBF). PIBF induces increased production of Th2-type cytokines; thus, it might stimulate antibody synthesis by B cells. There is a population of antibodies which, owing to the presence of a mannose-rich oligosaccharide residue on one of the Fab arms of the molecule, possess an asymmetric structure. Due to the asymmetric structure these molecules have no effector functions; however, they might act as blocking antibodies. This study was aimed at investigating the effect of progesterone-dependent immunomodulation on antibody production by B cells, with special emphasis on the synthesis of asymmetric nonprecipitating antibodies. The ratio of asymmetric IgG was significantly higher in supernatants of hybridoma cells cultured in the presence of PIBF than in those cultured in the absence of PIBF. Lymphocytes from healthy pregnant women produce significantly more PIBF than those of women with pathological pregnancies. The present studies revealed a positive relationship between asymmetric antibody content of the sera and PIBF expression on lymphocytes. Blocking of progesterone receptors by RU 486 or neutralizing endogenous PIBF activity by specific antibody significantly reduced the production of asymmetric antibodies in pregnant mice. Effector function of conventional and asymmetric antibodies was compared in a TNF alpha neutralization assay. Purified asymmetric anti-TNF alpha antibodies did not neutralize the cytotoxic effect of TNF alpha on L929 murine fibroblast target cells, whereas conventional anti-TNF alpha antibodies in the same concentration significantly (P < 0.001) reduced cytotoxicity. Our data suggest that PIBF induces increased production of asymmetric antibodies. These antibodies fail to exhibit effector functions and by blocking fetally derived antigens might contribute to protection of the fetus.

Inhibition by interleukin-1 beta of noradrenaline release in rat spleen: involvement of lymphocytes, NO and opioid receptors.

Effects of indomethacin, N omega-nitro-L-arginine (NNA) and naloxone, and of pretreatment with cyclophosphamide (CY), on the interleukin (IL)-1 beta induced inhibition of exocytotic noradrenaline release were investigated in the isolated, vascularly perfused spleen of the rat. Neurotransmitter release was evoked by perivascular electrical stimulation (4 Hz) and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection. Perfusion of the spleen with Tyrode's solution containing IL-1 beta (100 pg/ml) for 90 min caused an inhibition of the stimulation-evoked noradrenaline overflow which persisted for at least 20 min after washout of the IL. The evoked overflow was reduced in the presence of NNA 30 mumol/l, but remained unaffected by indomethacin 3 mumol/l, naloxone 0.1 mumol/l or treatment of the rats with CY (250 mg/kg). The opioid agonist etorphine 10 mumol/l inhibited the evoked overflow of noradrenaline and this effect was prevented by naloxone 0.1 mumol/l. The inhibition of evoked overflow by IL-1 beta was not affected by indomethacin but was reduced or even prevented in the presence of NNA or naloxone, or after lymphocyte depletion of spleens by CY. The results are compatible with the idea that in the rat spleen exocytotic noradrenaline release is accompanied by a concomitant secretion of a nitric oxide (NO)-like compound which, in turn, reinforces noradrenaline release, and that the release can be inhibited via prejunctional opioid receptors. The IL-1 beta induced inhibition of evoked release appears to be a complex process which involves as one of many steps a decrease of the facilitatory NO-like compound and the release of endogenous opioids probably from spleen lymphocytes.

Effects of human recombinant interleukins on stimulation-evoked noradrenaline overflow from the rat perfused spleen.

Experiments were carried out in the isolated spleen of the rat to study in a lymphoid organ the influence of interleukins (ILs) on noradrenaline release. Spleens were perfused with Tyrode's solution and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection. Perivascular electrical stimulation (4 or 10 Hz, 20-28 mA, 2 min) caused an increase in noradrenaline overflow and in perfusion pressure, both of which were markedly reduced by perfusion with Ca(2+)-free solution, abolished by tetrodotoxin, unaffected by hexamethonium, and subject to alpha 2-adrenoceptor- and muscarinic receptor-mediated modulation as shown by the effects of rauwolscine and methacholine. Human recombinant IL-1 beta and IL-2 and mouse recombinant IL-2 10 ng/ml failed to affect the evoked overflow of noradrenaline after an exposure time of 15 min. In contrast, human recombinant IL-1 beta and IL-2 0.1 ng/ml reduced the evoked overflow after exposure for 80 min; the inhibition tended to increase 30 min later despite washout. Murine recombinant IL-2 1.2 ng/ml caused no change after contact with the tissue for 80 min but there was an inhibition 30 min later after washout. Human recombinant IL-6 (0.1 ng/ml) caused no significant change. The inhibitory effect of low concentrations of IL-1 beta and IL-2 supports the idea that locally produced mediators of the immune system may affect neuronal function.

A muscarinic receptor different from the M1, M2, M3 and M4 subtypes mediates the contraction of the rabbit iris sphincter.

In order to analyse the subtype of muscarinic receptors involved in the methacholine-induced contraction of the rabbit iris sphincter we have determined equilibrium dissociation constants (KB) of various antagonists in the sphincter muscle. The values were compared with those observed at M1 (rabbit vas deferens), M2 (heteroreceptors in rat iris) and M3 receptors (guinea-pig ileum), or at the muscarinic receptors in the guinea-pig uterus. The methacholine-induced contraction of the uterus from immature guinea-pigs was competitively antagonized by pirenzepine (6.64, -log KB), 4-DAMP (8.39), hexahydrodifenidol (HHD; 7.00 for the (R)- and 5.40 for the (S)-enantiomer), p-fluoro-hexahydrosiladifenidol (p-F-HHSiD; 6.25) and valethamate bromide (8.04). The affinity of the antagonists is consistent with the presence of an M2 receptor. The -log KB values of the antagonists in the rabbit iris sphincter (6.43, p-F-HHSiD; 6.22, AQ-RA 741; 7.23 and 5.34, (R)- and (S)-trihexyphenidyl) were lower than, or within the lowest range of, estimates in the other experimental models, irrespective of the subtype selectivity of the antagonist. This excludes the presence of an M1, M2, M3 or M4 receptor in this smooth muscle. The affinity of UH-AH 37 in the iris was intermediate between that for M1 or M3, and M2 receptors. The low affinity of AQ-RA 741 and the low enantiomeric ratio of trihexyphenidyl (THP) in the iris (77.6) would be compatible with a presumed M5 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

[Intra-partum ethamsylate (dicynone) administration in the prevention of cerebral hemorrhage in premature infants]. / Intrapartum elkezdett ethamsylate (dicynone) kezelés a koraszülöttek agyvérzésének megelözésére.

Dicynone has been in use in all premature births prophylactically since 1987 by the authors. The administration of the drug begins before or during delivery. The diagnoses of cerebral haemorrhage was established on autopsy and the cases were compared with the previous years when Dicynone was not administered. During prophylactic use of Dicynone the cerebral haemorrhages significantly reduced among premature babies. It is well known, that the etiology of the cerebral haemorrhages are multifactorial. Their favourable experiences confirm the literary communications, whereas use of Dicynone can be one of the efficacious preventive drug against palsy of the premature babies.

Alpha-adrenoceptor mediated facilitation of acetylcholine release in the rat perfused heart.

Rat hearts were isolated with the vagus nerves intact and perfused, and the neuronal acetylcholine stores were pulse labeled with [14C]choline. The overflow of [14C]choline/acetylcholine evoked by extrinsic vagus nerve stimulation (3 Hz and 720 pulses or 10 Hz and 1200 pulses) was determined by liquid scintillation spectrometry and used as a measure for acetylcholine release. The postjunctional changes in atrial contraction and beating frequency were also recorded. Compared to controls, oxymetazoline and xylometazoline [but not clonidine, (3,4-dihydroxyphenyl-amino)-2-imidazoline), methoxamine and norepinephrine] enhanced the evoked overflow of 14C-activity in a concentration-dependent manner without changing the ratio between choline and acetylcholine determined by paper chromatography. Norepinephrine (10 mumol/l) inhibited the evoked overflow in the presence of propranolol plus yohimbine. The oxyme-tazoline-induced increase in evoked overflow was unaffected by rauwolscine (1 mumol/l), idazoxan (0.3 and 5 mumol/l), and yohimbine (0.3 and 5 mumol/l), but significantly reduced by phentolamine (5 mumol/l), prazosin (0.03 mumol/l), the (-)-enantiomer of WB 4101 (0.1 mumol/l) and SK&F 104078 (3 mumol/l). The overflow of 14C-activity evoked by field stimulation was increased by oxymetazoline the absence and presence of hemicholinium-3. The results are compatible with an alpha-1 adrenoceptor-mediated facilitation of exocytotic acetylcholine release from the rat heart in vitro. The increase in evoked neurotransmitter overflow, however, was not accompanied by an increase in postjunctional heart responses to vagus stimulation due to nonselective blocking properties of the facilitating agonists.

Different muscarinic receptors mediate autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat isolated perfused heart.

Experiments were carried out on rat isolated perfused hearts with both vagus nerves attached. The acetylcholine stores were labelled with [14C]-choline. The effects of muscarinic receptor antagonists on the [14C]-overflow and increase in perfusion pressure evoked by vagus nerve stimulation (10 Hz, 4-10 mA) were studied in order to determine the muscarinic receptor type involved in autoinhibition of acetylcholine release and vagally-induced vasoconstriction in the rat heart. Stimulation of the vagus nerves (1200 pulses) caused an increase in [14C]-overflow and in perfusion pressure which was significantly reduced by hexamethonium 500 mumol/l and abolished by tetrodotoxin 0.3 mumol/l or perfusion with Ca2(+)-free solution. The fractional rate of evoked [14C]-overflow per pulse upon stimulation at 10 Hz (720 pulses) was doubled in the presence of the non-selective antagonist atropine (0.01-1 mumol/l) as well as in that of the M2-selective compounds methoctramine (0.1 mumol/l) and AF-DX 116 (0.1-1 mumol/l), but remained unaffected by the M3-selective hexahydrosiladifenidol (0.1 mumol/l). The increase in perfusion pressure upon nerve stimulation was reduced by atropine (0.01 mumol/l) or hexahydrosiladifenidol (0.1 mumol/l) to approximately 50% and increased by about 50% in the presence of AF-DX 116 (0.1 mumol/l). The results show that the autoinhibition of acetylcholine release in the rat heart is mediated by M2 receptors. On the other hand, the increase in perfusion pressure upon vagus nerve stimulation is caused by a different muscarinic receptor, more sensitive to hexahydrosiladifenidol than to M2-selective antagonists.

Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris.

The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with 14C-choline. The stimulation-evoked 14C-overflow was doubled in the presence of atropine 0.1 mumol/l but unaffected by the agonist (+/-)-methacholine (50 mumol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mumol/l)-sensitive peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M2-selective antagonists methoctramine (10 mumol/l) and gallamine (100 mumol/l) increased both the 14C-overflow and the peak contractions evoked by field stimulation. In contrast, the M3-selective antagonist hexahydrosiladifenidol (0.1-10 mumol/l) failed to affect the evoked 14C-release but concentration-dependently (1-10 mumol/l) reduced the iris contractions. Pirenzepine (10 mumol/l) enhanced the evoked 14C-overflow and inhibited the peak contractions (0.1-10 mumol/l; maximal effect at 10 mumol/l). The low potency of the antagonist at both receptor sites indicates that an M1 muscarine receptor is not involved. The results are consistent with the idea of M2 muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M3-like receptors inducing the contraction of the sphincter muscle.

M2 muscarinic receptors on the iris sphincter muscle differ from those on iris noradrenergic nerves.

The pre- and postjunctional affinity constants of a series of muscarinic antagonists were determined in guinea pig and rabbit irises. Field stimulation-evoked [3H]noradrenaline release from superfused isolated irises was concentration dependently inhibited by (+/-)-methacholine, confirming the presence on the iris noradrenergic nerves of prejunctional inhibitory muscarinic receptors. The affinity constants of the antagonists at the pre- and postjunctional receptors are compatible with the coexistence in the iris of two different M2 receptors: the cardiac (M2 alpha) subtype on the noradrenergic nerves and the smooth muscle (M2 beta) subtype on the iris sphincter muscle. The rank order of potency of the antagonists studied at the prejunctional site was: atropine greater than himbacine greater than AF-DX 116 greater than pirenzepine greater than hexahydrosiladifenidol. The order of potency at the postjunctional receptors mediating the methacholine-induced isotonic contraction of the isolated rabbit iris sphincter was: atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than himbacine greater than AF-DX 116.

Muscarinic inhibition of [3H]-noradrenaline release on rabbit iris in vitro: effects of stimulation conditions on intrinsic activity of methacholine and pilocarpine.

1. Rabbit isolated irides were loaded with [3H]-noradrenaline and superfused with Tyrode solution. The inhibition by the muscarinic agonists (+/-)-methacholine and pilocarpine of the [3H]-noradrenaline overflow into the superfusate evoked by field stimulation (pulses of 1 ms duration, 75 mA) was measured as an index of activation of presynaptic muscarinic receptors. 2. The fractional rate of release per pulse during the first stimulation period (S1) was low with 360 pulses at 3 Hz, intermediate with 360 pulses at 10 Hz and high with 1200 pulses at 10 Hz. Upon repetitive stimulation (7 periods at 20 min intervals), the fractional rates of release per pulse during S7 no longer differed, suggesting a 'long-term' regulation of [3H]-noradrenaline release depending on the stimulation conditions. 3. The evoked [3H]-noradrenaline overflow was depressed by (+/-)-methacholine in a concentration-dependent manner. The EC50 ranged from 0.29 to 0.42 microM. Methacholine nearly abolished the transmitter release evoked at 3 Hz but reduced that induced at 10 Hz by only 50%. Under the latter condition the methacholine concentration-inhibition curve was bell-shaped and no muscarinic inhibition was observed in the presence of methacholine 30 microM. After washout of methacholine the evoked [3H]-noradrenaline release was temporarily enhanced. 4. Atropine 0.1 microM enhanced the [3H]-noradrenaline overflow (evoked by stimulation with 360 or 1200 pulses at 10 Hz), probably antagonizing a presynaptic inhibition by endogenous acetylcholine. The inhibition by methacholine was competitively antagonized by atropine 0.1 microM (apparent -log KB = 8.5-9.0). 5. Depending on the concentration, pilocarpine reduced the [3H]-noradrenaline overflow evoked by 360 pulses at 3 Hz up to 63%. However, at 10 Hz stimulation frequency the compound was inactive as an agonist but competitively antagonized the presynaptic inhibition induced by methacholine. The KB under the latter condition (0.95 microM) was very close to the EC50 value determined at 3 Hz (0.85 microM). 6. The results demonstrate a muscarinic inhibition of noradrenaline release from the rabbit isolated iris. The activation by pilocarpine of the presynaptic receptors provides an alternative explanation for the miosis induced in the rabbit in vivo, which might be the result of a decreased sympathetic tone in the iris dilator muscle.

Influence of a receptor reserve on the inhibition by calcium channel blockers of alpha adrenoceptor-mediated responses in rat isolated vascular tissues.

The effects of the Ca entry blockers nifedipine and verapamil on the contractions induced by phenylephrine (PHE) in rat isolated aorta and mesenteric vascular bed (MVB) were evaluated. The main goal was to elucidate whether differences among blood vessels in their receptor reserves are involved in the different degrees to which Ca antagonists inhibit alpha adrenergic vasoconstriction. In the two tissues the responses to the full agonist PHE were antagonized by prazosin with an at least 1000-fold greater potency than by yohimbine. The -log KB values for both antagonists in the aorta (11.07 and 7.40, respectively) were significantly higher than those in the MVB (9.77 and 6.43, respectively), thus suggesting receptor heterogeneity between the two tissues. Both nifedipine and verapamil were more effective in reducing the responses in the MVB (maximal inhibition, 54.3 +/- 1.9 and 55.0 +/- 3.5%) than in the aorta (25.2 +/- 5.8 and 30.4 +/- 0.7%). Studies with phenoxybenzamine (PB) indicated that in the latter case the responses were associated with an effective receptor reserve of about 40%, whereas in the MVB no spare receptors for the full agonist were available. Pretreatment of the MVB with PB showed no effect on the inhibitory action of verapamil. Conversely, removal of the spare receptors in the aorta (10(-10) M PB) rendered the responses more susceptible to inhibition by verapamil. Pretreatment of the aortic strips with 10(-9) M PB, however, failed to enhance further the effectiveness of verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha 1-Antagonist activity of the alpha 2-adrenoceptor agonist guanabenz in perfused mesenteric artery of the rat.

In the perfused mesenteric artery of the rat phenylephrine produced a concentration-dependent increase in perfusion pressure which was competitively antagonized by low concentrations of prazosin (pA2 9.56). Guanfacine, clonidine and guanabenz, three alpha 2-selective agonists, produced no response. However, guanabenz antagonized the phenylephrine-induced effect competitively (pA2 6.92) though guanfacine and clonidine did not affect it. These results suggest that the alpha 2-adrenergic agonist guanabenz possesses alpha 1-antagonist activity as well.