Effect of adenosine and adenosine analogues on cyclic AMP accumulation in cultured mesangial cells and isolated glomeruli of the rat. 1. Changes in intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were studied in rat isolated glomeruli and cultured glomerular mesangial cells exposed to adenosine and to the preferential A1 receptor agonist N6-R-1-methyl-2-phenylethyl adenosine (R-PIA), or the potent A2 adenosine receptor agonist 5-(N-ethylcarboxamide)adenosine (NECA). 2. Whereas NECA and adenosine triggered a dose-dependent increase in cyclic AMP values with EC50 values of approximately 10(-6) M and 3 x 10(-5) M respectively, R-PIA lowered cyclic AMP levels at concentrations of 10(-6) M or less and increased them at higher concentrations. 3. The time-course of the increase induced by 10(-6) M NECA was slower than that induced by 10(-4) M adenosine. Adenosine produced a maximal stimulation within the first minute, whereas the effect of NECA in both glomeruli and mesangial cells was noticeable only from the second minute of incubation. 4. The effects of the agonists R-PIA and NECA on the cyclic AMP system were blocked respectively by the A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthihe (DPCPX) at 10(-6) M and the A2 antagonist N-(2-dimethylaminoethyl)-N-methyl-4-(2, 3, 6, 7-tetrahydro-2,b-dioxo-1, 3-dipropyl-1H-purin-8-yl) benzene sulphonamide (PD115,199) at 10(-6) M. Theophylline, a known antagonist of adenosine receptors, inhibited the action of adenosine on cyclic AMP in mesangial cells. Dipyridamole, an inhibitor of the uptake of adenosine by the cells, enhanced the response to adenosine.5. These results suggest the existence of Al and A2 adenosine receptors with opposite actions on intracellular levels of cyclic AMP in both glomeruli and mesangial cells. Adenosine seems to increase cyclic AMP through the activation of a surface adenosine receptor with pharmacological properties distinct from those exhibited by the A2 adenosine receptor.


Inhibition of sympathetic hypertensive responses in the guinea-pig by prejunctional histamine H3-receptors. 1. The effect of (R)-alpha-methylhistamine, a selective H3-histamine receptor agonist, was examined on the neurogenic hypertension and tachycardia that is induced by stimulation of areas in the medulla oblongata of guinea-pigs. Electrical medullary stimulation (32 Hz, 3-5 s trains, 0.5-1.0 ms square pulse, 25-400 microA) produced intensity-dependent increases in blood pressure and a more variable tachycardia. 2. (R)-alpha-methylhistamine inhibited the hypertension and tachycardia due to submaximal CNS stimulation. The inhibition of hypertension by (R)-alpha-methylhistamine was dose-dependent (10-300 micrograms kg-1, i.v.) and was not seen at high intensities of stimulation. 3. (R)-alpha-methylhistamine (300 micrograms kg-1, i.v.) did not attenuate the pressor response to adrenaline (1 and 3 micrograms kg-1, i.v.), indicating that the effect of (R)-alpha-methylhistamine was not mediated by a postjunctional action on smooth muscle. 4. The inhibition of CNS-induced hypertension by (R)-alpha-methylhistamine (300 micrograms kg-1, i.v.) was blocked by the H3 antagonists, thioperamide (ID50 = 0.39 mg kg-1, i.v.), impromidine (ID50 = 0.22 mg kg-1, i.v.) and burimamide (ID50 = 6 mg kg-1, i.v.). The rank order potency of these antagonists is consistent with activity at the H3B receptor subtype. Chlorpheniramine (30 micrograms kg-1, i.v.) and cimetidine (3 mg kg-1, i.v.) did not antagonize the inhibition of CNS-hypertension by (R)-alpha-methylhistamine. 5. These results suggest that (R)-alpha-methylhistamine inhibits sympathetic hypertensive responses in guinea-pigs by activation of prejunctional H3-receptors, possibly located on postganglionic nerve terminals. Furthermore, on the basis of the rank order potency to different H3-antagonists, it appears that the H3B-receptor subtype is involved with H3-receptor responses on vascular sympathetic nerves.


Inhibition of noradrenaline release in the rat vena cava via prostanoid receptors of the EP3-subtype. 1. In segments of the rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution (containing desipramine and corticosterone), we studied the effects of prostaglandins of the D, E and F series, of a prostacyclin analogue and a thromboxane-mimetic and of subtype-selective prostaglandin E-receptor (EP-receptor) ligands on the electrically (0.66 Hz)-evoked tritium overflow. 2. The electrically-evoked tritium overflow was inhibited by prostaglandin E2 (maximum inhibition by about 80%; pIC40 7.49). The effect of prostaglandin E2 was not affected by rauwolscine, which, by itself, increased the evoked overflow; the alpha 2-adrenoceptor antagonist was added to the superfusion medium in all subsequent experiments. Indomethacin failed to affect either the evoked tritium overflow or its inhibition by prostaglandin E2. 3. The inhibitory effect of prostaglandin E2 on the electrically-evoked tritium overflow was not altered by the EP1-receptor antagonist. AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) at a concentration at least 30 fold higher than its pA2 value at EP1-receptors. The following compounds mimicked the effect of prostaglandin E2 showing the following rank order of potencies: misoprostol (EP2-/EP3-receptor agonist) congruent to sulprostone (EP1-/EP3-receptor agonist) congruent to prostaglandin E1 = prostaglandin E2 >> iloprost (EP1-/IP-receptor agonist) = prostaglandin F2 alpha. The evoked overflow was not affected by high concentrations of prostaglandin D2 or the thromboxane-mimetic U46619 (9,11-dideoxy-11 alpha, 9 alpha-epoxy-methano-prostaglandin F2 alpha). 4. The present results suggest that the postganglionic sympathetic nerve fibres innervating the rat vena cava are endowed with presynaptic EP3-receptors.They are not tonically activated by endogenously formed products of cyclo-oxygenase and do not interact with the presynaptic M2-adrenoceptors.


Long-term changes in gerbil brain neurotransmitter receptors following transient cerebral ischaemia. 1. Receptor autoradiographic and histological techniques were used to investigate long-term changes in the gerbil brain following transient cerebral ischaemia. 2. Transient ischaemia was induced for 3 min and 10 min, and the animals were allowed to survive for 8 months. 3. Histological examination revealed that 3 min ischaemia caused neuronal damage and mild shrinkage only in the hippocampal CA1 sector. Ten minutes of ischaemia produced severe neuronal damage in the striatum and the hippocampal CA1 and CA3 sectors. Considerable shrinkage was seen in the hippocampus; the dentate gyrus, however, was not damaged. 4. Three minutes of ischaemia produced changes in the binding of [3H]-quinuclidinylbenzilate ([3H]-QNB), [3H]-muscimol, and [3H]-MK-801 in various brain regions, as determined autoradiographically. In contrast, [3H]-cyclohexladenosine ([3H]-CHA) and [3H]-PN200-110 ([3H]-isradipine) binding in the brain was unaltered. 5. Ten minutes of ischaemia resulted in a major loss of neurotransmitter receptors, especially in the hippocampus. The substantia nigra showed a significant reduction in [3H]-CHA binding, whereas the striatum, which was morphologically damaged, showed no significant changes in any of the neurotransmitter receptors examined. 6. The results demonstrated that long-term survival after transient cerebral ischaemia produced alterations in neurotransmitter receptors, especially in the hippocampal formation, where considerable shrinkage was noted. These results also suggest that the hippocampal damage was not static, but progressive.


Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells. 1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.


A pertussis toxin-sensitive mechanism of endothelin action in porcine coronary artery smooth muscle. 1. Endothelin-1 (ET-1)-induced contraction of porcine coronary artery strips may be mediated via at least two intracellular signalling mechanisms, the activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels and the stimulation of phosphoinositide breakdown. Here we have investigated the possible involvement of pertussis toxin (PT)-sensitive guanosine-5'-triphosphate (GTP)-binding proteins (G-proteins) in ET-1-induced activation of these two signalling pathways in porcine coronary artery smooth muscle. 2. Increase in extracellular K+ concentration (10, 15 mM) shifted the dose-response relationship for the ET-1-induced contraction to the left. 3. The dihydropyridine Ca2+ channel blocker, nifedipine (10(-8) M), induced a rightward shift in the dose-response curve for ET-1. Pretreatment of the arterial strips with PT (0.1 microgram ml-1) induced a similar rightward shift of the ET-1 dose-response curve but not of the KCl response. Nifedipine (10(-8) M) did not further attenuate the ET-1-induced contraction in the PT-pretreated strips. 4. The pretreatment with PT significantly reduced 45Ca2+ uptake of the arterial strips stimulated by ET-1, but had no effect on ET-1-induced production of inositol phosphates. 5. The contractile response of the arterial strips to phorbol dibutyrate, an active phorbol ester, was not significantly affected by 10(-8) M nifedipine. 6. We confirmed that the pretreatment of the tissue with PT induced ADP-ribosylation of a 41 kDa membrane protein. 7. These findings indicate that activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels by ET-1 in this tissue is mediated via a PT-sensitive G-protein in a manner apparently independent of the ET-1-induced activation of protein kinase C. It is concluded that the action of ET-1 in porcine coronary artery is mediated via two distinct signal transduction pathways, which are coupled to PT-sensitive and PT-insensitive GTP-binding proteins, respectively.


Effects of noradrenaline on rat paratracheal neurones and localization of an endogenous source of noradrenaline. 1. Intracellular recording techniques were used to study the actions of exogenous noradrenaline (NA) on rat paratracheal neurones in situ. The receptor subtypes underlying these actions were investigated by application of selective adrenoceptor antagonists. 2. Application of NA (0.1-10 microM) by superfusion evoked a membrane depolarization in 85% (52 out of 61) of all paratracheal neurones studied. The response consisted of a slow depolarization which was sometimes accompanied by action potential discharge. In 26 out of 31 cells the response was associated with a change in input resistance of the cell membrane. In 22 out of 26 cells there was a 30% increase, whilst in a further 4 cells there was a 15% decrease in input resistance. The amplitude of the NA depolarization was concentration-dependent. 3. The depolarization evoked by NA was reversibly antagonized by prazosin (1 microM) but unaffected by yohimbine (1 microM) or propranolol (1-10 microM). 4. High performance liquid chromatography with electrochemical detection (h.p.l.c.-e.c.d.) was used to assay for NA and dopamine in samples containing mainly paratracheal ganglia and in samples of tracheal smooth muscle with mucosa. NA was present in all samples assayed at a level of 1.6 micrograms NA g-1 and 0.5 microgram NA g-1 wet weight of the two sample types respectively. Dopamine was not detected in any samples of either ganglia or smooth muscle with mucosa. 5. It is concluded that NA-evoked depolarizations of rat paratracheal neurones result from stimulation of alpha 1-adrenoceptors, and that local levels of NA may be sufficiently high to activate these receptors directly.


Pharmacological modulation of inhaled sodium metabisulphite-induced airway microvascular leakage and bronchoconstriction in the guinea-pig. 1. We have investigated the effects of chlorpheniramine, atropine and capsaicin pretreatment on inhaled sodium metabisulphite (MBS)-induced airway microvascular leakage and bronchoconstriction in anaesthetized guinea-pigs in order to clarify the mechanisms involved in these responses. The effects of frusemide and nedocromil sodium were also examined. 2. Lung resistance (RL) was measured for 6 min after inhalation of MBS (20, 40, 80 and 200 mM; 30 breaths), followed by measurement of extravasation of Evans blue dye into airway tissues, used as an index of airway microvascular leakage. MBS caused an increase in RL and leakage of dye at all airway levels in a dose-dependent manner. 3. Chlorpheniramine (10 mg kg-1, i.v.), atropine (1 mg kg-1, i.v.), their combination or inhaled nedocromil sodium (10 mg ml-1, 7 min) had no effect against the airway microvascular leakage induced by 80 mM MBS (30 breaths). Capsaicin pretreatment (50 mg kg-1, s.c.) caused a significant decrease in the leakage of dye in the main bronchi and inhaled frusemide (10 mg ml-1, 7 min) also in the main bronchi and proximal intrapulmonary airway. 4. Chlorpheniramine, atropine, their combination, capsaicin pretreatment and frusemide, but not nedocromil sodium, inhibited significantly the peak RL induced by 80 mM MBS (30 breaths) by approximately 50%. 5. We conclude that a cholinergic reflex and neuropeptides released from sensory nerve endings may participate in the mechanisms of MBS-induced airway responses. Frusemide but not nedocromil sodium may have an inhibitor effect on these neural mechanisms. The inhibitory effect of nedocromil sodium against lower doses of MBS is not excluded.


Involvement of multiple receptors in the biological effects of calcitonin gene-related peptide and amylin in rat and guinea-pig preparations. 1. The activity of rat alpha and beta calcitonin gene-related peptide (CGRP) as compared to the structurally related peptide, rat amylin, has been investigated in the guinea-pig isolated left atrium (electrically driven), in mucosa-free strips from the base of the guinea-pig urinary bladder and in the rat isolated vas deferens (pars prostatica). The antagonist activity of the C-terminal fragment of human alpha CGRP, alpha CGRP(8-37), was also investigated. 2. In the guinea-pig isolated left atrium the three peptides produced a concentration-related positive inotropic effect, amylin being about 16 and 31 times less potent than alpha or beta CGRP, respectively. Human alpha CGRP(8-37) produced a rightward displacement of the log concentration-response curve to the three agonists tested, without depression of maximal response attainable. Apparent pKB values calculated on the basis of the displacement produced by 1 microM human alpha CGRP(8-37) indicated an agonist-independent affinity of the antagonist (6.66 +/- 0.11 for alpha CGRP, 6.42 +/- 0.17 for beta CGRP and 6.95 +/- 0.11 for amylin). 3. In the guinea-pig isolated urinary bladder, alpha or beta CGRP or amylin produce a concentration-related inhibition of twitch contractions evoked by train electrical field stimulation (10 Hz frequency, 0.25 ms duration at 100 V for 0.5 s every 60 s). Amylin was about 100 times less potent than alpha or beta CGRP. Human alpha CGRP(8-37) (3 microM) did not significantly affect the inhibitory action of the three agonists tested.4. In the rat isolated vas deferens, a or PCGRP or amylin produced a concentration-related inhibition of twitch contractions evoked by electrical field stimulation (0.2 Hz frequency, 0.5 ms duration at 60 volts). Amylin was about 100 times less potent than a or PCGRP. Human aCGRP(8-37) at 3 microM did not significantly affect the inhibitory action of amylin and at 3 microM antagonized the responses to rat a and PCGRP with apparent pKB values of 5.86 +/- 0.15 and 6.11 +/- 0.13, respectively.5. These findings indicate that multiple receptors mediate the actions of peptides of the CGRP/amylin family in the preparations investigated. In the guinea-pig atrium both a and P forms of rat CGRP as well as amylin act by stimulating a single class of receptors which are sensitive to the inhibitory action of human aCGRP(8-37). In rat isolated vas deferens, at least two receptors could be present, one activated by a and PCGRP and partially sensitive to human aCGRP(8-37) and another which is sensitive to amylin but not recognised by human aCGRP(8-37). This latter type of receptor could be entirely responsible for the action of the agonists in the guinea-pig urinary bladder.


Changes in intrinsic inhibition in isolated hippocampal slices during ethanol withdrawal; lack of correlation with withdrawal hyperexcitability. 1. Intracellular recordings were made from pyramidal cells in area CA1 in mouse isolated hippocampal slices, after chronic ethanol treatment in vivo. 2. Fast i.p.s.ps were isolated by injection of the impaled neurones with QX314 (to block fast sodium currents and the slow i.p.s.p.) and stimulating the interneurones in the presence of the glutamatergic blockers, CNQX and APV. 3. The isolated fast-inhibitory postsynaptic potential (f.-i.p.s.p.) was measured at intervals during the 7 h withdrawal period. The reversal potential and sensitivity to bicuculline suggested that the isolated f.-i.p.s.p. was mediated by activation of the GABAA receptor-chloride ionophore complex. 4. Measurement of stimulus-response relationships for the f.-i.p.s.ps revealed an initial increase in the maximum size of the i.p.s.p., evoked from a membrane potential of -50 mV, seen at 2 h into ethanol withdrawal. This was attributed to a negative shift in the reversal potential, Ei.p.s.p., with no observed change in conductance, Gi.p.s.p. 5. No differences in f.-i.p.s.ps evoked during ethanol withdrawal or in control slices were seen at 4 h or 6 h. At these times, epileptiform activity was seen in previous field potential recordings. 6. Paired pulse depression of the f.-i.p.s.p. was significantly increased at 2 h into withdrawal, when a 150 ms pulse interval was used. No differences were seen at later times in the ethanol withdrawal period. 7. The results suggest that ethanol withdrawal hyperexcitability in isolated hippocampal slices is not caused by primary decreases in inhibition mediated by the GABAA receptor-chloride ionophore complex.4. Measurement of stimulus-response relationships for the f.-i.p.s.ps revealed an initial increase in the maximum size of the i.p.s.p., evoked from a membrane potential of - 50 mV, seen at 2 h into ethanol withdrawal. This was attributed to a negative shift in the reversal potential, Ejp.sp with no observed change in conductance, Gj ps p.5. No differences in f.-i.p.s.ps evoked during ethanol withdrawal or in control slices were seen at 4 h or 6 h. At these times, epileptiform activity was seen in previous field potential recordings.6. Paired pulse depression of the f.-i.p.s.p. was significantly increased at 2 h into withdrawal, when a 150 ms pulse interval was used. No differences were seen at later times in the ethanol withdrawal period.7. The results suggest that ethanol withdrawal hyperexcitability in isolated hippocampal slices is not caused by primary decreases in inhibition mediated by the GABAA receptor-chloride ionophore complex.The increase in the f.-i.p.s.p. during the initial stages of the withdrawal might prevent the overt expression of epileptiform activity at this time.


Millimolar amiloride concentrations block K conductance in proximal tubular cells. 1. Amiloride, applied at millimolar concentrations, results in the blockade of K+ conductance in amphibian proximal convoluted cells (PCT), fused into giant cells. 2. Amiloride results directly in a blockade of K+ conductance that is not related to inhibition of the Na(+)-H+ antiport, which would lower intracellular pH, adversely affecting K+ conductance. On the contrary, high amiloride concentrations promote entry of this lipophilic base in the cell, leading to higher cell pH. 3. Under voltage clamp conditions, control vs. amiloride, current-voltage curves from PCT fused giant cells intersect at -86.2 +/- 3.4 mV, a value close to the equilibrium potential for potassium. 4. Hexamethylene amiloride, 10(-5) M, irreversibly depolarizes the membrane potential. 5. Barium decreased by 50% the initial slope of realkalinization, following removal of a solution containing NH4Cl, as did amiloride. In addition, these blockers reduced membrane conductance by 40%, suggesting that a fraction of the amiloride-suppressible NH4+ efflux may be conductive. 6. Amiloride does not directly inhibit the Na(+)-K+, ATPase in our preparation, contrary to the prevalent belief. 7. In vivo studies show that amiloride interferes with an apical K+ conductance but it does not alter basolateral K+ conductance.


Agonist analysis of 2-(carboxycyclopropyl)glycine isomers for cloned metabotropic glutamate receptor subtypes expressed in Chinese hamster ovary cells. 1. 2-(Carboxycyclopropyl)glycines (CCGs) are conformationally restricted glutamate analogues and consist of eight isomers including L- and D-forms. The agonist potencies and selectivities of these compounds for metabotropic glutamate receptors (mGluRs) were studied by examining their effects on the signal transduction of representative mGluR1, mGluR2 and mGluR4 subtypes in Chinese hamster ovary cells expressing the individual cloned receptors. 2. Two extended isomers of L-CCG, L-CCG-I and L-CCG-II, effectively stimulated phosphatidylinositol hydrolysis in mGluR1-expressing cells. The rank order of potencies of these compounds was L-glutamate > L-CCG-I > L-CCG-II. 3. L-CCG-I and L-CCG-II were effective in inhibiting the forskolin-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in mGluR2-expressing cells. Particularly, L-CCG-I was a potent agonist for mGluR2 with an EC50 value of 3 x 10(-7) M, which was more than an order of potency greater than that of L-glutamate. 4. L-CCG-I evoked an inhibition of the forskolin-stimulated cyclic AMP production characteristic of mGluR4 with a potency comparable to L-glutamate. 5. In contrast to the above compounds, the other CCG isomers showed no appreciable effects on the signal transduction involved in the three mGluR subtypes. 6. This investigation demonstrates not only the importance of a particular isomeric structure of CCGs in the interaction with the mGluRs but also a clear receptor subtype specificity for the CCG-receptor interaction, and indicates that the CCG isomers would serve as useful agonists for investigation of functions of the mGluR family.


Possible mechanisms of inhibition with atropine against noradrenaline-induced contraction in the rabbit aorta. 1. In the rabbit isolated aorta, atropine (3 x 10(-6) M-10(-4) M) inhibited contractile response to noradrenaline without affecting contraction to KCl. 2. In the presence of contraction to noradrenaline, atropine (3 x 10(-7) M-10(-4) M) caused concentration-dependent relaxation. Pretreatment with theophylline (10(-3) M) potentiated the relaxant action of atropine. Relaxation to atropine was not affected by the specific guanosine 3':5'-cyclic monophosphate phosphodiesterase inhibitor, M & B 22,948 (10(-4) M), tetraethylammonium (10 mM), indomethacin (10(-5) M), propranolol (10(-7) M), nifedipine (10(-6) M) or removal of the endothelium. 3. Relaxation to either atropine or prazosin was not affected by preincubation with prazosin and atropine, respectively. 4. In Ca(2+)-free medium containing EGTA and nifedipine, atropine (10(-7) M-10(-4) M) inhibited the residual noradrenaline response more than the subsequent Ca(2+)-induced contraction. Pretreatment with either theophylline (10(-3) M), forskolin (3 x 10(-7) M) or a low concentration of prazosin (3 x 10(-9) M) also inhibited the residual contraction to noradrenaline and Ca2+. The effect of combined treatment of atropine and any of these agents was much greater than with each individual agent. 5. Atropine (10(-6) M-10(-4) M) also inhibited increases in the level of inositol monophosphates (IP) in response to noradrenaline. Theophylline (10(-3) M) and a low concentration of prazosin (3 x 10(-9) M) also inhibited IP formation. Combined with atropine, the effect was much greater than with each of these agents individually. 6. Atropine did not affect adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the aorta and also failed to displace specific [3H]-prazosin binding.7. These results suggest the possibility that smooth muscle relaxation to atropine may be due to the inhibition of phosphoinositide metabolism. The relaxation is not apparently due to an action of atropine on ax-adrenoceptors, or a change in the level of cyclic AMP.


Effects of propofol and enflurane on action potentials, membrane currents and contraction of guinea-pig isolated ventricular myocytes. 1. The effects of two general anaesthetics, propofol and enflurane, on electrical activity and contractions were investigated in single myocytes isolated from guinea-pig ventricles. 2. Propofol and enflurane depressed the plateau and shortened the duration of action potentials. 3. Under voltage-clamp conditions, propofol and enflurane reduced the amplitude of inward calcium current and of additional inward current activated by cytosolic calcium. 4. Contractions (measured with an optical technique) accompanying either action potentials or second inward currents (in response to depolarizations to 0 mV) were reduced by both anaesthetics. The mechanisms for calcium entry during contractions accompanying pulses to positive potentials such as +60 mV are thought to differ from those accompanying second inward currents which are evoked by pulses from -40 to 0 mV. Enflurane enhanced the amplitudes of contractions accompanying pulses to positive potentials; in contrast these contractions were depressed by propofol. 5. In experiments where recovery processes were investigated by use of pairs of voltage-clamp pulses with a variable interval between them, enflurane but not propofol slowed the recovery of contractions and calcium-activated 'tail' currents. These observations are consistent with the hypothesis that enflurane may impair calcium handling by the sarcoplasmic reticulum whereas propofol has little, if any, effect at this site. 6. In conclusion, the actions of propofol and enflurane on second inward currents contribute to their effects on action potentials and contraction. The negative inotropic effect of both anaesthetics may result partly from reduced calcium influx to trigger contraction, and for enflurane, partly from an impairment of calcium handling by the sarcoplasmic reticulum.


Effects of the delta-opioid receptor antagonist naltrindole on antinociceptive responses to selective delta-agonists in post-weanling rats. 1. Antagonism, by the selective delta-opioid receptor antagonist naltrindole, of the antinociceptive effects of [D-Pen2, D-Pen5] enkephalin (DPDPE), [D-Ser2, Leu5, Thr6] enkephalin (DSLET) and D-Ala2 deltorphin I (DELT I) has been studied in 25 day old rats. 2. Antinociception was measured by the 50 degrees C tail immersion test following i.p. administration of agonists and/or antagonists. 3. Dose-related antinociception was observed with DPDPE, DSLET and DELT I and ED75 doses were computed (0.66 mg kg-1, 0.65 mg kg-1, 0.032 mg kg-1 respectively) and used for antagonism studies. 4. Naltrindole (0.01 mg kg-1) significantly attenuated the antinociceptive effects of DPDPE and DSLET with 0.1 mg kg-1 producing complete reversal of the effects of the ED75 dose. In contrast, naltrindole at 0.01 and 0.1 mg kg-1 did not alter antinociceptive responses to DELT I. Naltrindole at 1 mg kg-1 significantly attenuated DELT I antinociception. 5. Naloxone (1 mg kg-1) produced equivalent degrees of antagonism of the antinociceptive effects of DPDPE, DSLET and DELT I. ICI 174,864 (1 mg kg-1) also antagonized antinociception with a differential degree of attenuation (DSLET > DPDPE > DELT I). 6. Naltrindole (1 mg kg-1) had no effect on the antinociception induced by the selective mu-agonist alfentanil (60 micrograms kg-1). Naltrindole, naloxone or ICI 174,864 had no effect on nociceptive latencies. 7. The differential antagonism by naltrindole of the effects of three selective delta-agonists suggests delta-receptor heterogeneity.Further, the lower sensitivity of response to DELT I suggests that this agent may exert its antinociceptive effects at a different 6 receptor subtype from DPDPE or DSLET.


Lack of effect of the antimigraine drugs, sumatriptan, ergotamine and dihydroergotamine on arteriovenous anastomotic shunting in the dura mater of the pig. 1. In anaesthetized animals, the antimigraine drugs, sumatriptan, ergotamine and dihydroergotamine, reduce carotid arteriovenous anastomotic shunting. Within the carotid vascular bed arteriovenous anastomoses are located, amongst other places in the dura mater, which is a putative site of the pain during a migraine attack. 2. In this investigation, we have localized and measured the arteriovenous shunting within the carotid vascular bed of the pig by using simultaneous intracarotid injections of radiolabelled microspheres of three different sizes (10, 15 and 50 microns), which provides an index of blood flow via arteriovenous anastomoses larger than approximately 14, 27 and 90 microns diameter, respectively. The effects of sumatriptan (0.3 mg kg-1), ergotamine (0.02 mg kg-1), dihydroergotamine (0.1 mg kg-1) and saline were studied by repeating the injections of 15 and 50 microns spheres after the treatments. 3. There was no difference in shunting or entrapment between the 10 and 15 microns microsphere, indicating the absence of arteriovenous anastomoses with a diameter between 14 and 27 microns. 4. Arteriovenous anastomoses with a diameter between 27 and 90 microns, as indicated by the difference in blood flow measured by 15 and 50 microns spheres, were located in the dura mater, ears, skin, fat and, to a lesser extent, in the skeletal muscles and eyes. 5. Sumatriptan, ergotamine and dihydroergotamine reduced the overall flow in the smaller arteriovenous anastomoses (diameter between 27 and 90 microns), and even more in larger shunts (wider than 90 microns). 6. Locally, blood flow in the smaller arteriovenous shunts was reduced in the skin and fat, but not in the dura mater, ears, eyes and muscles.It is not possible to determine in which tissues blood flow in the larger arteriovenous anastomoses was reduced.7. Tissue blood flow measured with 15 gm microspheres remained unchanged after the three antimigraine drugs, implying a lack of effect on capillary flow.8. It is concluded that in the anaesthetized pigs the only evident effect of these antimigraine drugs on carotid haemodynamics is a decrease in blood flow in both smaller and larger arteriovenous anastomoses;the smaller arteriovenous anastomoses were affected in the skin and fat, but not in other tissues.


Differential potentiation of GABAA receptor function by two stereoisomers of diimidazoquinazoline analogues. 1. U-84935, diimidazo[1,5-a;1',2'-C]quinazoline,5-(5-cyclopropyl-1,2,4-oxid iazol-3yl)- 2,3-dihydro, is a ligand of high affinity for the benzodiazepine site of the GABAA receptor composed of alpha 1 beta 2 gamma 2 subunits. 2. The efficacy of its analogues was measured with their ability to potentiate GABA-mediated Cl- currents in the whole cell configuration of the patch clamp techniques in human kidney cells (A293 cells) expressing the subtype of the GABAA receptor. 3. The analogues displayed various levels of efficacy including agonists, partial agonists and antagonists without marked changes in their affinity for the receptors. 4. The major determinant of their efficacy was the spacial configuration of a methyl substituent of the C2 atom of the rigid and planar diimidazoquinazoline ring: U-90167, containing the methyl substituent projected below the plane of the ring, markedly enhanced the GABA current with a maximal potentiation of 220 +/- 25%, while its stereoisomer, U-90168, marginally increased the GABA response with a maximal potentiation of 45 +/- 10%, to which its methyl group appeared to contribute very little. 5. U-90167 potentiated the GABA response with an EC50 of 8.1 nM and a Hill coefficient of 1.1 and did not alter the reversal potential for the Cl- current. 6. From computational modelling, the sensitive methyl group of U-90167 could be assigned to the general region for the 5-phenyl group of diazepam. The diimidazoquinazoline, because of its rigid and plantar ring structure, may be useful to define further the out-of-plane region responsible for agonistic activity and to pinpoint other areas pivotal to the functionality of benzodiazepine ligands.


The effect of centrally acting myorelaxants on NMDA receptor-mediated synaptic transmission in the immature rat spinal cord in vitro. 1. The effect of the myorelaxant drugs baclofen, diazepam and tizanidine have been compared on in vitro preparations of baby rat spinal cord and adult rat superior cervical ganglion. 2. Dorsal root-elicited long duration (time to half decay 9.71 +/- 0.29 s.e. mean, n = 31) ipsilateral ventral root reflexes (DR-VRP), measured as integrated area, of immature rat spinal cord preparations were abolished by RS-2-amino-5-phosphonopentanoate (AP5) (EC50 8.13 +/- 0.92 microM, n = 3). The initial short latency component of DR-VRP was resistant to AP5. 3. Baclofen abolished both components of the DR-VRP. Respective EC50 values for the AP5-insensitive and AP5-sensitive components were 237 +/- 68 nM (n +/- 7) and 57 +/- 10 nM (n = 7). These effects of baclofen were reversed by the GABAB antagonist, CGP35348. The apparent Kd values (16.7 +/- 6.4 microM, n = 3 and 14.3 +/- 3.9 microM, n = 6 respectively) for this reversal were not significantly different. 4. Tizanidine, clonidine and diazepam had no effect on the AP5-insensitive component of the DR-VRP. 5. The AP5-sensitive long duration component of the DR-VRP was depressed to respective maximal levels of 23.2 +/- 1.4% (n = 7), 18.8 +/- 3.8% (n = 4) and 47.6 +/- 1.6% (n = 5) of control (100%) levels by tizanidine (EC50 135 +/- 33 nM), clonidine (EC50 26.0 +/- 2.2 nM) and diazepam (EC25 114 +/- 12 nM, n = 4). The depressant effects of tizanidine and clonidine were reversed by idazoxan (1 microM). Flumazenil (I microM) failed to reverse the depressant effect of tizanidine. The depressant effect of diazepam was reversed by flumazenil (1 microM) but not by idazoxan (1 microM). Naloxone 1 M did not reverse the effects of either tizanidine or diazepam.6. In the presence of tetrodotoxin (0.1 SAM) which abolished synaptic activity, clonidine, tizanidine or diazepam (10, 100 and 101JM respectively) produced no significant antagonism of NMDA-induced depolarizations recorded from ventral roots.7. Control (100%) synaptic responses of rat superior cervical ganglion preparations were depressed respectively to near maximal levels of 60.0 +/- 5.2% (n = 4) and 60.7 +/- 5.6% (n = 5) by clonidine (0.5 JAM,EC25 15.3 +/- 3.0 nM) and tizanidine (1 JAM, EC25 227 +/- 83 nM). These depressant effects were reversed by idazoxan (1 AM). Baclofen (EC25 28.7 +/- 10.0, n = 3) depressed the postganglionic response to a maximum level of 71.8 + 2.4% (n = 4) control at a concentration of 100 microM. The latter depressant action was reversed by the GABAB receptor antagonist, CGP35348 (1 mM). Diazepam (1 microM) had no significant effect on ganglionic transmission.8. It is concluded that the activation of benzodiazepine or M2-noradrenaline receptors can modulate NMDA receptor-mediated excitatory synaptic pathways whereas synaptic excitation from primary afferent terminals, mediated by non-NMDA receptors, appears to lack the propensity for this type of modulation. The results show also that the isolated spinal preparation can be used to identify central myorelaxant actions that are mediated through functional benzodiazepine or X2-noradrenaline receptors.


Multiple self-damaging behaviour among alcoholic women. A prevalence study. Among patients being treated for specific behaviour-control problems, there exists an important subgroup of 'multi-impulsive' patients whose treatment might be facilitated if the full range of their problems were recognised and dealt with as one general issue of impulse control. In women in particular, loss of control of eating may be prevalent and easily concealed from staff, and may thwart treatment. This survey of 50 women attending an alcoholic-treatment unit explored the prevalence of behavioural-control problems other than those of alcohol. Three-quarters of the women also had other behavioural problems. Over half the sample had thought of taking an overdose and just under half had actually taken one; about a quarter had cut themselves deliberately; half described impulsive physical violence; half acknowledged a period of 'promiscuity'; and at least 16% had had a clinically diagnosable eating disorder. More research is needed but we believe that all self-damaging behaviour should be addressed simultaneously to prevent 'revolving door' relapses as emotional distress is transferred from one behaviour to another.


Staging of hilar cholangiocarcinoma by ultrasound and duplex sonography: a comparison with angiography and operative findings. The pre-operative radiological assessment of proximal bile duct tumours is clinically important as resection may be limited by tumour extension along the bile ducts, into hepatic parenchyma or the adjacent vascular structures. Demonstration of the extent of biliary and vascular involvement can direct additional investigations and definitive treatment. 22 patients with hilar cholangiocarcinoma were studied pre-operatively by conventional ultrasound (US) and duplex sonography (DS). The extent of tumour infiltration and vascular involvement was compared with arteriography and operative findings. Bile duct dilatation and the level of obstruction was documented by US in 22 (100%), and the tumour was shown by US in 19 (86%). In these 19 patients, the extent of extraductal extension compared with operative findings was correct in 13, underestimated in two, and in four infiltration was massed. Vascular patency or involvement was correctly determined by DS in 19 (86%), and by arteriography in 18 (82%). In two of the three incorrect DS interpretations, lobar atrophy and contralateral hypertrophy distorted the hilar anatomy. US with DS is valuable in the pre-operative staging of proximal bile duct tumours in predicting ductal and vascular involvement.


Adult Wilms' tumour: review of 14 patients. Wilms' tumour, or nephroblastoma, is the commonest renal neoplasm found in children, but is rarely found in adults, the world literature recording only approximately 200 cases. Individual case reports continue to be published but only within the last 10 years have definitive treatment regimes been suggested. In order to determine the UK experience of adult Wilms' tumour, members of the British Association of Urological Surgeons were circulated with a questionnaire, and from 141 replies, 13 members reported 17 cases. Critical review of the original histology slides excluded 3 of these; the 14 remaining cases are described and their management discussed in the light of current recommended treatment.


Clinicopathological study of nephroblastomas in Ibadan. The present study reviews 31 patients with histologically confirmed nephroblastoma seen over a 5-year period (1985-1989). There was a predominance of female patients with a male: female ratio of 1:1.7, and the average age of the patients was 47 months. The patients invariably presented with a palpable abdominal mass, but haematuria was exceptional. The neoplasms tended to be larger on average than those reported previously among Caucasian children. Carcinomatous transformation of the epithelial components was observed in 2 renal neoplasms. Poorly differentiated neoplasms were commoner in male than in female patients. Our findings indicate an unfavourable prognosis for nephroblastoma in this environment because of late clinical presentation with advanced disease and inadequate treatment and follow-up.


Diagnostic and therapeutic aspects of fine-wire localization biopsy for impalpable breast cancer. During the first 2 years (July 1989 to July 1991) of the Avon Breast Screening Service, fine-wire localization biopsy was indicated in 213 impalpable breast lesions. A total of 144 lesions were benign and 69 malignant. Only four of 213 lesions (1.9 per cent) were not excised at the first localization. Factors influencing reoperation in the 69 patients with malignant impalpable lesions were examined. There was a significant association (P < 0.001) between parenchymal disturbances on mammography and invasive carcinoma, and between non-invasive carcinoma and microcalcification (P < 0.001). In 31 patients the localization biopsy was the only surgical procedure. Thirty-eight patients required further surgery: 12 underwent further local excision and 26 mastectomy. Reoperation was more frequent in patients with calcification than in those with parenchymal disturbance (P < 0.001). The most frequent indications for mastectomy were inadequate excision of widespread comedo ductal carcinoma in situ or invasive ductal carcinoma combined with extensive ductal carcinoma in situ. Fine-wire localization biopsy was a combined therapeutic and diagnostic procedure in 31 of 69 women with impalpable screen-detected lesions. The majority of patients required further surgery because radiological abnormalities underestimated the extent of disease.


Survival after hepatic resection for malignant tumours. A retrospective analysis of 194 patients who underwent hepatic resection for primary or metastatic malignant disease from January 1962 to December 1988 was undertaken to determine variables that might aid the selection of patients for hepatic resection. Hepatic metastases were the indication for resection in 126 patients. The 5-year survival rate was 17 per cent. For patients with resected metastases from colorectal cancer (n = 104), the survival rate at 5 years was 18 per cent. The 5-year survival rate was 27 per cent when the resection margin was > 5 mm compared with 9 per cent when the margin was < or = 5 mm (P < 0.01). No patient with extrahepatic invasion, lymphatic spread, involvement of the resection margin or gross residual disease survived to 5 years, compared with a 23 per cent 5-year survival rate for patients undergoing curative resection (P < 0.02). The survival rate of patients with poorly differentiated primary tumours was nil at 3 years compared with a 20 per cent 5-year survival rate for patients with well or moderately differentiated tumours (P not significant). The site and Dukes' classification of the primary tumour, the sex and preoperative carcinoembryonic antigen level of the patient, and the number and size of hepatic metastases did not affect the prognosis. The 5-year survival rate for patients with hepatocellular carcinoma (n = 42) was 25 per cent. An improved survival rate was found for patients whose alpha-fetoprotein level was normal (37 per cent at 5 years) compared with those having a raised level (nil at 3 years) (P < 0.01). Involvement of the resection margin, extrahepatic spread and spread to regional lymph nodes were associated with an 8 per cent 5-year survival rate versus 44 per cent for curative resection (P < 0.005). The presence of cirrhosis, the presence of symptoms, and the multiplicity and size of the tumour did not affect the prognosis. The 5-year survival rate of 11 patients with hepatic sarcoma was 25 per cent. No patient with peripheral cholangiocarcinoma survived to 1 year in contrast to patients with hilar cholangiocarcinoma, all four of whom survived for more than 14 months.


Biodistribution of Lipiodol following hepatic arterial injection. Thirteen patients undergoing selective coeliac angiography before insertion of an indwelling hepatic arterial cannula underwent injection of 3 ml radiolabelled Lipiodol (2 MBq 131I) into the hepatic artery at the end of the procedure. At subsequent laparotomy 1-9 days later, biopsies were taken from normal liver and metastases. The radioactivity of this material was measured to establish the tumour:liver ratios. Two patients with large metastases (> 10 cm in diameter) had low ratios. In the remainder, the median ratio at 24 h was 1.5:1 (range 1.1-2.5:1; n = 5) and 2.6:1 (range 1.5-64.0:1; n = 6) at 3-9 days. Four patients underwent single photon emission computed tomography, which confirmed selective retention of Lipiodol in small metastases, although no activity was detected in a large deposit (> 15 cm) 10 days after injection. The tumour:liver ratio in the other three patients increased from 3.0-5.6:1 on day 1 to 4.5-7.2:1 on day 6. This study suggests that Lipiodol may be a useful therapeutic delivery agent to small colorectal liver metastases.


Familial amyloid polyneuropathy associated with the transthyretin Cys114 gene in a Japanese kindred. A Japanese kindred with dominantly inherited amyloid polyneuropathy, commonly called familial amyloid polyneuropathy (FAP), has been identified. Amyloid protein was transthyretin (TTR) related and the patients were heterozygous for the mutant gene encoding TTR with a single amino acid substitution of cysteine for tyrosine at position 114. This family originated in Nagasaki Prefecture, Japan, and 12 of the 36 known members of six generations have been affected. The initial symptoms occurred in their thirties with the cardinal features of polyneuropathy, vitreous opacities and cardiac disease. Sensory neuropathy was severe in the lower limbs. Autonomic disturbances, especially postural hypotension, were the most debilitating to the patients. Amyloid deposits were detected widely in most organs except for the central nervous system. The duration from the onset of the disease to death was within 10 yrs. Heart failure caused by heavy amyloid deposits was the most common cause of sudden death.


The role of heterarchical control in the evolution of central pattern generators. The acceptance of the concept of central pattern generators (CPGs) led to the perception that descending inputs initiate stereotyped movements, such as locomotion, but play relatively minor roles after the movement begins. Sensory input could entrain the CPG, and the CPG was responsive to the proper inputs for switching, etc. Evidence is here presented that the influences of both descending and sensory inputs are two-way. Descending inputs are shown to be involved in an ongoing manner during locomotion, as it has been found that CPGs are phasically driving the same descending systems that themselves activate the CPGs. Similarly, sensory inputs are being actively processed by the CPG and, here again, produce a two-way interaction between sensory input and CPGs. Finally, mechanical factors are shown to be major contributors to the form of the movement. Thus, overall the CPG can only be considered as one of several contributors to any movement; all concurrently process the flow of information. Control is viewed as distributed, that is, as heterarchical as opposed to hierarchical. Because of the complexity of interaction between the levels of the system, it is argued that any change in the body will propagate through the system and effect the final output regardless of where the change originates. Thus, ontogenetic and phylogenetic changes must have influences felt throughout the entire system. Examples are presented to demonstrate that this is, indeed, the case. The effect of these changes will primarily be manifest in the interface between the layers, that is, in the interface between the descending and sensory inputs and the CPG, so that the changes can be adaptively accommodated on a moment by moment basis when necessary.


The application of biotechnology to the control of foot-and-mouth disease virus. Biotechnology, which less than 10 years ago was heralded as an alternative to epidemiology in providing the answers to the control of foot-and-mouth disease (FMD), has not fulfilled its initial promise. Instead it is now complementing epidemiology by providing extremely sensitive and specific tools for identifying and characterizing strains of FMD virus in diagnostic material. Considerable advances in our understanding of the evolution of the virus in different field situations has been made possible by the development and application of polymerase chain reaction and nucleotide sequencing techniques. The individual genes of FMD virus can now be cloned into a number of vectors and separately expressed and studied in isolation from the other viral proteins. Biotechnology has not provided a safe and effective vaccine to replace the conventional tissue culture derived vaccine that would have made FMD a disease of the past. FMD remains the most economically significant animal disease having a major influence on the international trade of animals and their products. The world distribution of FMD has remained almost unchanged over the last 20 years and a balance has been maintained between improved surveillance and diagnostic technology and the ever increasing legal and illegal international movement of animals and reduction in veterinary resources. Research continues on peptide, recombinant and vector expressed virus protein vaccines which could at any time yield a breakthrough, not only for FMD control but, using similar technology, for control of other viral diseases, human and animal. Until this occurs, control and eradication of FMD still relies on classical epidemiological techniques, making use of new biotechnological methods.


Cognitive enhancing properties of beta-CCM infused into the nucleus basalis magnocellularis of the rat. Peripheral administration of various benzodiazepine derivatives or beta-carbolines (inverse agonists at benzodiazepine receptors), has been shown to affect memory. In this study, the effect of local infusion of a beta-carboline-methyl beta carboline-3-carboxylate (beta-CCM) into the nucleus basalis magnocellularis (NBM) of rats was examined in a two-trial recognition task. The results show that beta-CMM (3 micrograms/0.5 microliter) enhances recognition performance when injected both before or immediately after the acquisition trial. These effects appear to be mediated by a benzodiazepine (BZD) receptor since they were blocked by pretreatment with Ro 15-1788, a BZD receptor antagonist. This study supports the involvement of the NBM in cognitive processes, and demonstrates that these processes can be influenced by alteration of GABAergic neurotransmission.


Brain corticotropin-releasing hormone receptors on neurons and astrocytes. Corticotropin-releasing hormone (CRH) exerts many potent effects within brain and is considered an important brain neuroregulator. CRH acts via receptors that are widely distributed throughout brain which exhibits highest CRH receptor concentrations in extrahypothalamic regions. We have previously characterized CRH receptors in heterogeneous extrahypothalamic forebrain cell cultures consisting of neurons and glia, and have shown them to exhibit similar kinetic and pharmacological characteristics as CRH receptors in pituitary and in situ brain. However, it is not known whether CRH receptors are present on neurons, glia or both. We tested the hypothesis that CRH receptors are present on neurons in extrahypothalamic forebrain cell cultures derived from day 17-18 fetal rats by characterizing receptors in predominantly neuronal (N), glial/astrocytic (G) cultures and mixed (M) cultures. Mean CRH receptor concentrations (fmol/mg protein) in N (10.4), G (9.4), and M (9.8) cultures were similar. Following Scatchard analyses derived from competition curves, all cell populations exhibited similar mean high-affinity/low-capacity (Kd = 1.0-1.9 nM; Bmax = 183-388 fmol/mg protein) and low-affinity/high-capacity (Kd = 92-104 nM; Bmax = 2034-5008 fmol/mg protein) classes of binding sites. (1) Neurons and astrocytes in fetal extrahypothalamic brain cell cultures contain CRH receptors which exhibit similar concentrations and similar kinetic characteristics. (2) These observations suggest that biological effects of CRH in brain could be mediated via actions on neurons and/or glial astrocytes.


Localization and characterization of binding sites for vasopressin and oxytocin in the brain of the guinea pig. Using autoradiography on film, specific binding sites for arginine-vasopressin (AVP) and for oxytocin (OT) were localized in various areas of the brain of adult male guinea pigs. Vasopressin binding sites were detected with [3H]AVP or with [125I]VPA, a recently synthetized linear vasopressin antagonist radiolabeled with 125I. [125I]VPA and [3H]AVP yielded similar results, thus suggesting that AVP binding sites present in the guinea pig brain are V1 type receptors. Supporting evidence on this was obtained in competing studies using structural analogues allowing to discriminate V1 receptors from V2 and from OT receptors. Oxytocin binding sites were labeled with [3H]OT or with the iodinated OT antagonist [125I]OTA; both ligands yielded similar results. The localization in the guinea pig brain of AVP binding sites differed from that of OT binding sites. AVP binding sites were mainly detected in the olfactory bulb and throughout the cerebral cortex. Oxytocin binding sites were most noticeable in the hypothalamic ventromedial nucleus, in the amygdaloid complex and in restricted areas of the cerebral cortex. A comparison of the present data with those previously described in the rat, the mouse, the human and the hamster brain suggests that similar binding sites are present in these species, but that their anatomical distribution differs markedly. These data are discussed in relation to immunocytochemical and electrophysiological data which suggest that binding sites detected by autoradiography may represent, at least in part, functional neuronal receptors.


Feline immunodeficiency virus: a brief review. Feline immunodeficiency virus (FIV), previously known as feline T-lymphotropic lentivirus (FTLV), was first described by Pedersen et al. (1987) who isolated the virus from cats with a variety of clinical signs suggestive of immunodeficiency. Since then FIV has become one of the most studied feline viruses, not least because of its similarity to human immunodeficiency viruses (HIV) which cause acquired immunodeficiency syndrome (AIDS) in man.


The cocaine-induced elevation of plasma corticosterone is mediated by endogenous corticotropin-releasing factor (CRF) in rats. The role of endogenous corticotropin-releasing factor (CRF) in the cocaine-induced corticosterone response was investigated by using the immunoneutralization and receptor blockade of endogenous CRF. Pretreatment with different dilutions (1:5, 1:10 and 1:20, i.c.v.) of CRF antibody and different doses of an antagonist for CRF receptors, alpha-helical CRF9-41 (alpha h-CRF, 0.001-1.0 micrograms, i.c.v.), dose-dependently prevented the cocaine-induced increase in corticosterone level. These results support the hypothesis that the activation of the hypothalamo-pituitary-adrenal (HPA) axis by cocaine is mediated through the release of endogenous CRF.


Regulation of flunitrazepam binding in the dorsal horn of the spinal cord by adrenalectomy and corticosteroids. Adrenal corticosteroids and adrenalectomy (ADX) have opposing effects on benzodiazepine binding sites in brain regions. These treatments were employed to study [3H]flunitrazepam (FLU) binding in regions punched out from the rat spinal cord. We found that binding was higher in dorsal horn than in ventral horn, and minimal in white matter. Clonazepam and RO 15-1788 largely displaced [3H]FLU binding, whereas RO 5-4864 was weakly active. Four days post-ADX, binding increased exclusively in the dorsal horn, and this effect was reversed by administration of corticosterone (CORT), but not dexamethasone (DEX) or aldosterone (ALDO) given over 4 days. When endogenous CORT was increased by administration of cold stress to adrenal-intact rats, reduced benzodiazepine (BDZ) binding was also observed in the dorsal horn. When added in vitro, only ALDO and not CORT or DEX, inhibited [3H]FLU binding. It is suggested that steroids with affinity for the type I corticosteroid receptor (CORT, ALDO) decrease [3H]FLU binding to a neural-type BDZ receptor in the dorsal horn. Reduction of the inhibitory BDZ system may be physiologically important, and can partly explain the enhancement of excitatory synaptic transmission produced by corticosteroids at the level of the spinal cord.


Regulation of neurotransmitter enzyme by quisqualate subtype glutamate receptors in cultured cerebellar and hippocampal neurons. The possible involvement of ionotropic and metabotropic quisqualate (QA) receptors in neuronal plasticity was studied in cultured glutamatergic cerebellar or hippocampal cells in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When cerebellar or hippocampal neurons were treated with QA, it elevated the specific activity of glutaminase in a dose-dependent manner. The half-maximal effect was obtained at about 0.1 microM, the maximum increase was at about 1 microM, but levels higher than 10 microM QA produced progressive reduction in glutaminase activity. In contrast, QA had little effects on the activities of lactate dehydrogenase and aspartate aminotransferase and the amount of protein, indicating that the increase in glutaminase was relatively specific. The QA-mediated increase in glutaminase was mimicked by the ionotropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; EC50, about 0.5 microM), but not by the metabotropic QA receptor agonist trans-(+-)-1-amino-cyclopentyl-1,3,dicarboxylate (t-ACPD; up to 0.5 mM). The specific ionotropic QA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited QA- and AMPA-mediated increases in glutaminase activity in a dose-dependent manner, whereas other glutamate receptor antagonists, D,L-2-amino-5-phosphonovalerate, gamma-D-glutamyl aminomethyl sulphonic acid and gamma-D-glutamyl diethyl ester were ineffective. The elevation of neurotransmitter enzyme was Ca(2+)-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)


Localization of the ras-like rab3A protein in the adult rat brain. Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e., dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, regions of cerebral cortex and the cerebellum exhibited striking laminar distributions of rab3A immunoreactivity. In the hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentate granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral cortical hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.


Zucker obese rats: defect in brain histamine control of feeding. Manipulation of hypothalamic histamine produced different effects on feeding between the Zucker obese (fa/fa) and their lean littermate rats (Fa/-). Infusion of a histamine H1-receptor antagonist into the third cerebroventricle elicited feeding in the lean and Wistar King A rats, but it did not affect feeding in the obese rats. To enhance hypothalamic neuronal histamine, thioperamide, and H3-receptor antagonist, was similarly infused. The lean and Wistar rats decreased their food intake after the infusion, but thioperamide produced no significant effect on feeding in the obese rats. Infusion of histamine into the third cerebroventricle mimicked the effects of thioperamide on feeding: reduction of food intake in the lean and Wistar rats, but no significant change in the obese rats. Hypothalamic histamine of the obese rats (0.430 nmol/g) was significantly lower than the lean (1.209 nmol/g) and Wistar rats (4.838 nmol/g). The histamine concentration of the cerebral cortex in the obese rats was also lower than the non-obese animals. The results indicate that the feeding abnormality of Zucker obese rats may be at least due to disturbance of histamine suppressive signals both at presynaptic and postsynaptic levels.


Characterization of the oligosaccharide side chains on kainate binding proteins and AMPA receptors. The amino acid sequences of the kainate binding proteins (KBPs) from frog and chicken brain are homologous with the carboxy terminal half of the rat brain AMPA receptors. In this study, we have characterized the oligosaccharide side chains present on the KBPs from chicken and frog brain, and the AMPA receptors (GluR1, GluR2, and GluR3) from rat brain. Deglycosylation of the asparagine-linked carbohydrates present on the chicken, frog, and rat receptor subunits with N-glycanase, resulted in decreases in the relative molecular weights (M(r)) of 3.4, 3.4, and 5.1 kDa respectively. Thus the percent of asparagine linked carbohydrate (based on M(r) values derived from SDS polyacrylamide gels) of the 49 kDa chicken, the 48 kDa frog, and the 107 kDa receptor rat subunits is 6.9, 7.1, and 4.8 percent respectively. No shifts in the M(r) were detected after treatment with neuraminidase indicating that sialic acid does not appear to be a major component of these receptors. Lectin binding studies demonstrated that both asparagine-linked and serine/threonine-linked oligosaccharides were present in the chicken, frog, and rat proteins. The data indicate that at least one of the asparagine linked oligosaccharide side chains appear to be of the complex or non-bisected hybrid type in all three species. The similarities in the glycosyl moieties of the chicken and frog kainate KBPs and the rat brain AMPA receptors suggests that the homology in the amino acid sequences between these proteins may extend to homology in their oligosaccharide sides chains as well.


Tau and ubiquitin in the human hypothalamus in aging and Alzheimer's disease. Immunocytochemical staining of hypothalamic cell groups with four antibodies to Alzheimer paired helical filaments (PHF) (i.e., anti-PHF serum 60e and monoclonal antibody (mAb) Alz-50, both directed against normal and abnormally phosphorylated tau; mAb tau-1, which recognizes tau; and mAb 3-39 to PHF, which recognizes the carboxy terminal domain of ubiquitin) revealed a clear distinction between 12 Alzheimer's disease (AD) patients and seven controls in the hypothalamus. Dystrophic neurites, which appeared to be the most specific components in AD, were most conspicuous after Alz-50 staining. However, Alz-50 also stained neuronal cytoplasm and normal, thin, beaded neurites in the paraventricular nucleus (PVN) of controls, even of young cases. This staining was clearly distinct from the staining of cytoplasm and dystrophic neurites in the PVN of Alzheimer patients. The abundant staining of dystrophic neurites and cell bodies in the nucleus tuberalis lateralis (NTL) in AD, in which no neuronal loss is observed, suggests that alterations in cytoskeletal markers do not necessarily indicate impending cell death. Moreover, the cytoskeletal changes in the NTL, sexually dimorphic and suprachiasmatic nuclei in AD indicate that this condition is not restricted to cortical areas or nuclei projecting to the cortex. Consequently, the pathophysiological implications of cytoskeletal staining in AD are at present far from clear. The human hypothalamus may not only provide a better insight into the pathogenesis of Alzheimer's disease, but could also be of help in the neuropathological diagnosis of this condition.


GABA, acting at both GABAA and GABAB receptors, inhibits the release of cholecystokinin-like material from the rat spinal cord in vitro. Superfusion of slices of the dorsal zone of the lumbar enlargement of the rat spinal cord with an artificial cerebrospinal fluid allowed the collection of cholecystokinin-like material (CCKLM) whose Ca(2+)-dependent release could be evoked by tissue depolarization with 30 mM K+. Studies on the possible influence of GABA and related agonists on this process showed that the amino acid, the GABAA agonist, muscimol, and the GABAB agonist, baclofen, inhibited the K(+)-evoked release of CCKLM from the rat spinal cord in a concentration-dependent manner. Maximal inhibition did not exceed -40% with either agonist. Furthermore, the effects of GABAA and GABAB receptor stimulation were not additive. Whereas the effects of muscimol (10 microM) and baclofen (1 microM) could be completely antagonized by bicuculline (1 microM) and phaclofen (10 microM), respectively, complete blockade of the inhibition by GABA (1 microM) could only be achieved in the presence of both antagonists. These data indicate that both GABAA and GABAB receptors are involved in the negative influence of GABA onto CCK-containing neurones within the dorsal horn of the rat spinal cord. Apparently, these receptors are not located on CCK-containing neurones themselves, since the inhibitory effect of GABA on the K(+)-evoked release of CCKLM could be completely prevented by tetrodotoxin (1 microM). As CCK acts centrally as an endogenous opioid antagonist, such a GABA-inhibitory control of spinal CCK-containing neurones might participate in the analgesic action of the amino acid via the intrathecal route.


Alpha 1-adrenergic stimulation of low Km GTPase in rat striata is diminished with age. The effect of norepinephrine and epinephrine on the activity of low Km GTPase was studied in striata from young and old rats. Results showed that the amines stimulated the low Km GTPase activity of both young and old animals in a concentration-dependent manner. There was no differential effect of the amines as a function of age, but the stimulated low Km GTPase activity was significantly lower in the aged tissue.


F11 neuroblastoma x DRG neuron hybrid cells express inhibitory mu- and delta-opioid receptors which increase voltage-dependent K+ currents upon activation. The F11 cell line is a fusion product of cells of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal-root ganglion (DRG) neurons. Previous biochemical results suggest that they express mu- and delta-opioid receptors that are negatively coupled to adenylate cyclase. The present study provides direct agonist-binding and electrophysiologic evidence of mu and delta, but not kappa, receptor expression in F11 cells. Radioligand binding assays show that F11 cell membranes bind the mu- and delta-opioid receptor agonists, DAGO and DPDPE with Kd = 4.5 and 4.9 nM and Bmax = 111 and 195 fmol/mg, respectively. Tight-seal patch-clamp recordings of F11 cells after several days in a differentiating culture medium (low serum, cyclic AMP and nerve growth factor) showed that: (i) the outward K+ current during pulsed depolarization in most of these cells was increased by either DAGO or DPDPE, but none were responsive to both opioids or to the kappa-opioid receptor agonist, U-50,488H. The response was blocked by relevant receptor antagonists, naloxone, beta-funaltrexamine or naltrindole; (ii) cells without processes responded neither to DAGO nor to DPDPE; (iii) treatment with pertussis toxin blocked all opioid-induced increases in outward K+ current. The opioid-induced increase in voltage-dependent membrane K+ current in F11 cells resembles the inhibitory effect elicited by mu- and delta-opioid agonists in primary cultures of mouse DRG neurons.


Regional distribution of cells expressing glycine receptor alpha 2 subunit mRNA in the rat brain. The alpha 2 subunit of the glycine receptor is expressed transiently in the rat brain during early development suggesting that this subunit may be replaced by the alpha 1 subunit in the adult brain. The expression of glycine receptor alpha 2 subunit mRNA was investigated in the 7-day-old rat brain by in situ hybridization histochemistry using oligonucleotide probes specific for this subunit. Neurons expressing alpha 2 subunit mRNA were found to be widely and abundantly distributed throughout brain. We compared the distribution of neurons expressing alpha 2 subunit mRNA with that of neurons expressing alpha 1 or beta subunit mRNA. In the lower brainstem, the location of the neurons expressing alpha 2 subunit mRNA was very similar to that of the neurons with alpha 1 or beta subunit mRNA. Neurons expressing beta subunit mRNA were widespread and numerous in the forebrain, where neurons with alpha 1 subunit mRNA were uncommon. The locations of the neurons labeled by the alpha 2 probe were very similar to those of the cells labeled by the beta probe. These findings suggest that the alpha 2 subunit is not only expressed by immature neurons containing the alpha 1 subunit, but is also common to most immature neurons having the glycine receptor. However, it should be noted that several neurons contained beta and/or alpha 1 subunit mRNA but lacked alpha 2 subunit mRNA, suggesting that the glycine receptor is heterogeneous in its composition during brain development.


Induction of tolerance to ischemia: alterations in second-messenger systems in the gerbil hippocampus. Preconditioning the brain with sublethal ischemia protects against neuronal damage following subsequent ischemic insult. Using [3H]inositol 1,4,5-triphosphate (IP3), [3H]phorbol 12,13-dibutyrate (PDBu), [3H]cyclic adenosine monophosphate (cAMP) and [3H]rolipram, we performed quantitative autoradiography to determine postischemic alterations in second-messenger systems in the gerbil hippocampus following preconditioning the brain with sublethal ischemia. At 7 days of reperfusion, no alterations were observed in brains subjected to 2 min of forebrain ischemia which produced no neuronal damage. However, 3-min ischemia caused a 75% reduction in [3H]IP3 binding (p < 0.01 vs. control) and 15-25% reductions in [3H]forskolin (p < 0.01 vs. control), [3H]cAMP (p < 0.05 vs. control), and [3H]rolipram (p < 0.01 vs. control) binding in the CA1 subfield coincident with histopathological CA1 pyramidal cell destruction, but no significant alterations in [3H]PDBu binding. Preconditioning the brain with 2 min of ischemia followed by 4 days of reperfusion prevented both histopathological cell death and the reductions in binding following subsequent 3 min of ischemia. Interestingly, [3H]IP3 and [3H]rolipram binding in CA1 showed a transient reduction, by 30% and 20% (both p < 0.01 vs. control), respectively, in the early reperfusion period. This downregulation of the IP3 system may play a role in the protection against cell death.


Na+ current kinetics are not the determinants of the action potential duration in neurons of the rat ventral tegmental area. Na+ currents were recorded from two morphological subpopulations of neurons acutely dissociated from the rat ventral tegmental area (VTA). About 45% of 56 VTA cells examined possessed only the ordinary type of Na+ current which was blocked by a low concentration (0.2 microM) of TTX. However, the remaining 55% had the Na+ current which contained a small fraction of the TTX-insensitive component, irrespective of morphological variations and action potential durations of VTA cells. The peak amplitude of the TTX-insensitive component was less than 10% of the peak amplitude of the total Na+ current. The activation and inactivation kinetics of the TTX-insensitive component were much the same as those of the overwhelming TTX-sensitive component of the Na+ current in VTA cells but differed from those of the TTX-insensitive Na+ current reported in peripheral sensory neurons. Thus, it was concluded that the well-known different action potential durations found for subpopulations of VTA cells are not due to multiplicity of Na+ channel kinetics.


[Tinnitus: peripheral and central aspects]. Tinnitus is often related to a peripheral impairment which seems to trigger an abnormal autonomous functioning of the central auditory system. Moreover peripheral stimulations may diminish, and even suppress their perception, either through masking or through inhibition of an aberrant neuronal activity. Here we present an experimental model of peripheral deafferentation in which a decrease of neurochemical activity is observed in the brainstem auditory nuclei, followed by a progressive recovery after the peripheral destruction. This activity could represent a central morphofunctional correlate of tinnitus. Development of experimental models of tinnitus must be continued. Such models should allow the study of the basic physiological mechanisms behind tinnitus and suggest some therapeutic approaches which could be objectively evaluated.


[Chemoprophylaxis and chemotherapy of common colds caused by rhinoviruses: overview and outlook]. Rhinoviruses are the main etiologic agents of infectious common colds, which represent about 40% of the acute respiratory infections in man. The antigenic diversity of rhinoviruses precludes any prevention by vaccination. Within the last 20 years, efforts have therefore concentrated on chemoprophylaxis or chemotherapy with antiviral agents. Interferons (alpha and beta) administered intranasally at high doses, exerted a significant prophylactic activity in volunteers inoculated with rhinoviruses or in the course of epidemics in families. By contrast, the therapeutic activity of interferons is almost nonexistent, which greatly limits their practical interest. Various synthetic chemicals exert a marked and selective inhibitory effect on the replication of various serotypes of rhinoviruses in cell cultures. In the absence of an animal model of rhinovirus infection, in vivo studies of these molecules have been performed in human volunteers, chiefly at the Common Cold Research Unit in Salisbury (Great Britain). 3 synthetic compounds (an imidazothiazole, a benzimidazole derivative and a piperazinyl-pyridazine) have exerted a significant prophylactic activity, especially marked with the latter. None of these compounds, however, was active when administered after the infectious challenge. The search for selective anti-rhinovirus compounds is still going on; it will probably be facilitated through a combination of structural studies of rhinovirus capsids (or of their cellular receptors) with computer-assisted design of synthetic molecules. The recent observation of the influence of psychological factors on the propensity of rhinovirus-infected subjects to develop clinically apparent common cold must be taken into account in future studies.


Two homologous mitochondrial introns from closely related Saccharomyces species differ by only a few amino acid replacements in their Open Reading Frames: one is mobile, the other is not. We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis. The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns). We have shown that the S. capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S. cerevisiae intron is not. Determination of sequence of the S. capensis intron demonstrates that it differs from that of the homologous S. cerevisiae intron (bi2) by a very small number of nucleotide substitutions.


[Effects of zinc overdose on the viscera and tissue pathomorphology]. This investigation included two toxic animal models of zinc: acute and subacute. In the subacute model, ultrastructural changes occurred and zinc content of the liver and kidney increased when the concentration of serum zinc was not obviously changed. In the acute model, the functions of the liver, kidney and cell immunity were impaired, and marked ultrastructural changes noted. The results showed that long-term use of zinc or overdose is dangerous, although zinc is considered of lower toxicity as compared to other heavy metal elements. It is suggested that zinc should be used carefully in children.


[Color Doppler flow imaging in gallbladder tumors]. Thirty patients (22 male, 8 female, age range 25-83) with gallbladder (GB) masses or thickened GB wall found by real-time sonography were studied by duplex and color doppler ultrasonography. All the patients were confirmed pathologically after surgery. Seven of 8 patients with primary GB malignancy (adenocarcinoma 7, malignant fibrous histiocytoma 1) showed high velocity arterial blood flow signal (VABFS) in the tumor masses. No VABFS was observed in 10 patients (metastatic adenocarcinoma 3, adenoma 5, polyps 2). All the 10 patients with primary GB carcinoma (masses 8, wall thickening 2) showed high VABFS in the GB wall. In contrast, no VABFS was found in the GB wall in 3 patients with metastatic GB carcinoma. In 17 patients with benign GB lesion (adenoma 5, polyps 2, xanthogranuloma 2, chronic cholecystitis 6, subacute cholecystitis 2), only 5 (polyps 1, xanthogranuloma 1, chronic cholecystitis 1, subacute cholecystitis 2) demonstrated low VABFS. High VABFS in the GB masses or GB wall, a significant feature of primary GB carcinoma, is helpful in differentiating primary GB carcinoma from metastatic and benign GB lesion.


[Targeting therapy with adriamycin-monoclonal antibody (HAb18) conjugate in nude mice with human hepatocellular carcinoma xenografts]. Adriamycin (ADM) was linked covalently to the murine monoclonal antibody (McAb) HAb18 against human hepatocellular carcinoma via a dextran T-40 bridge. The molar ratio of HAb18: DEX: ADM was 1:2.4:56 in the conjugate. It was confirmed by immunochemical staining and ELISA that the antigen binding capacity of HAb18 in the conjugate was well preserved. In vitro study indicated that the conjugate cytotoxicity to tumor target cell lines was selective. The radio-immunoimaging and distribution of the conjugate labelled with 125I in the nude mice bearing human hepatocellular carcinoma showed that 125I-HAb 18-DEX-ADM conjugate was localized in the tumor site. The HAb18-DEX-ADM targeting therapy in nude mice bearing human hepatocellular carcinoma showed the following results: decreased tumor size and partly necrosed cancer tissue in contrast to the rapidly growing tumor in the control group (P < 0.05). Our study suggested that targeting therapy with HAb18-DEX-ADM conjugate may be useful in the treatment of patients with hepatocellular carcinoma.


[Immuno-ricin A chain specific for gastric cancer: internalization and its significance]. Biotinylated ricin A chain (RTA) was introduced to antigastric cancer monoclonal antibody MGb2 with chemical conjugating technique to form an immuno-ricin A chain conjugate (I-RTA) specific for gastric cancer. Its internalization in human gastric cancer cell line SGC-7901 was followed by double dynamic EM labelling procedure using streptavidin-gold and sheep anti-mouse IgG-gold probes. The effects of verapamil on I-RTA internalization routings were also observed. The results demonstrated that the main entry modality of I-RTA was non-coated microinvagination, followed by coated pits and interioration of microvilli. The internalized I-RTA was found to be quickly transported to multivesicular bodies and finally to lysosomes, where it was degraded, whilst in the non-membranous structures I-RTA was only occasionally encountered. Ultrastructurally, verapamil displayed its effects by making the internalized I-RTA stay longer in tubulovesicular structures and be delivered in a less amount and more slowly into lysosomes. Furthermore, I-RTA was increased in the non-membranous structures, and the in vitro killing effect was significantly enhanced. The present data revealed the basic cell biology process of I-RTA-cell interaction, which will provide not only an important clue to improving I-RTA targeting therapy efficiency, but also a new way for further screening targeting potentiators.


[Tumor necrosis factor alpha activity in peripheral blood in patients with acute leukemia: changes and clinical implications]. The production of tumor necrosis factor alpha (TNF alpha) of peripheral blood mononuclear cells was investigated in 58 patients with acute leukemia. The results showed that the production of TNF alpha was significantly lower in 41 of the 58 patients than that of normal controls. The TNF alpha activity levels from patients with acute monocytic leukemia and acute myelomonocytic leukemia were higher than those from patients with acute lymphoblastic leukemia and acute granulocytic leukemia (P < 0.05). The TNF alpha activity level changed in various courses of the disease and returned to normal during the period of complete remission. It was noted that patients with higher or normal TNF alpha levels had a higher complete remission rate than those with lower TNF alpha level (P < 0.01). These results indicated that determination of TNF alpha maybe helpful in observing changes of the disease and predicting prognosis.


[Immunological status of alveolar macrophages in patients with lung cancer and benign pulmonary diseases]. This is a report of the study on the immunological status of alveolar macrophages (aM phi) in patients with lung cancer (LC, n = 27) and benign pulmonary diseases (BD, n = 26). Patients were undergone bronchoalveolar lavage by fiberoptic bronchoscopy. aM phi in the lavage fluid isolated by adherence on plastic surface were examined in vitro for their cytostatic and cytolytic activities against tumor target cells, secretion of interleukin 1 (IL-1) and tumor necrosis factor (TNF), intracellular IL-1 activity and mRNA expression of IL-1 beta and TNF-alpha. aM phi, both non-activated and activated, were shown to be highly cytostatic against P815 cells by 3H-TdR post-labelling assay. There was no statistical difference between the LC and BD group. As shown by isotope release assay, regardless of being activated or not, aM phi were not cytolytic against P815 and NS-1 cells in both groups of patients. TNF activity could be demonstrated in the culture supernatants of aM stimulated with LPS. Statistically, the TNF activity was not different in the two groups of patients. Spontaneous release of TNF activity was occasionally detected in unstimulated aM phi. While both intracellular and extracellular IL-1 activity of unstimulated aM phi was demonstrated in the two patient groups, the former activity was 1 to 5 times as high as the latter. When stimulated with LPS, there was some increase in extracellular but not intracellular IL-1 activity. mRNA expression of IL-1 beta and TNF-alpha by dot blot hybridization was demonstrable in aM phi from both patient groups irrespective of activation. These results indicate that the immune status of aM phi in lung cancer patients examined does not differ from that in patients with benign pulmonary diseases.


[Epidemiology of birth defects among children in 8 provinces in China]. In 1988, birth defects (BD) among 85,170 children aged 0-14 were investigated in eight provinces and cities of China. The overall prevalence rate was 15.13% in urban, 15.40% in rural areas. The prevalence rate of BD was significantly higher in males than in females (16.66% vs 13.70%). The prevalence rate of BD was significantly higher in mothers aged > or = 35 years than in mothers aged < 35. 20 of 82 categories of BD accounted for > or = 1% of all BD. The 3 leading BD were: cutis angioma, congenital heart disease and indirect hernia. The incidence of mental retardation was significantly higher in children with BD than in children without BD.


[Mechanism of renal vein thrombosis in patients with nephrotic syndrome: a prospective study]. One hundred nephrotic patients were routinely examined with renal venography and renal biopsy. Blood platelet (BPC), aggregation (Pag), adhesiveness (Pad), kaolin prothrombin time (KPTT), thrombin time (TT), plasma viscosity (PV) were tested in 50 patients. Also plasma fibrinogen (Fbg), fibrin/fibrinogen degradation product (FDP), antithrombin III (AT III), antiplasmin (PI), plasminogen (Plg) were investigated from the peripheral vein, superior, inferior veno cava, and right, left renal vein. Forty-six nephrotic patients had the complication of renal vein thrombosis (RVT); 4/5 of them were clinically latent. Multivariate regression analysis, showed that the number of 2 renal branches with RVT (RVTn) related to the history of steroid and membranopathy was associated with the tendency of RVT formation. Coagulation-fibrinolysis test showed RVTn = 1.17284 Pag-0.02363 PI + 0.04034 AT III + 3.21141 PV-0.02447 Plg + 2.33538 (P = 0.025). Fbg in renal vein involved with RVT was significantly different from the innocent side (P < 0.05). In addition, correlations were found between AT III and protein in urine (P < 0.01) and between plasma Fbg and albumin (P < 0.02). 46% patients with nephrotic syndrome complicated by RVT were usually clinically latent. Disorders of platelet function, increased PV, steroid and intensive diuretic treatment might promote RVT formation, especially in membranopathy.


[Protective action of phenytoin on cerebral ischemia in rats]. In order to study and modify the ischemic brain lesions, protecting the reversible damage of neurons, and limiting the ischemic damage both models of cerebral ischemia--focal cerebral ischemia induced by photochemically, and brain reperfusion after ligation of common carotid arteries bilateral in Wistar rats were used to investigate the protective effect of diphenylhydantoin (DPH) on cerebral ischemia. Two groups of rats received DPH 10 mg/kg and 30 mg/kg respectively were compared with those having received normal saline immediately after cerebral ischemia. The effects of DPH on the changes of EEG, biochemical marker and pathologic lesion in focal cerebral ischemia in rats were evaluated. The results showed that the degree of restoration of EEG in the group treated with DPH was better than in the group with normal saline, the content of water in ischemic cerebral tissue was decreased significantly (P < 0.05), the activity of Na-K-ATPase and antioxidant was increased significantly (P < 0.01, P < 0.05, respectively), and the percentage of necrotic neurons in periischemic area was decreased markedly (P < 0.001) in groups treated by DPH. The results suggest that there is a definite protective effect of DPH on cerebral ischemia.


[Tetrandrine on monocyte migration induced by endothelial cell-derived chemotactic factor]. The medium conditioned by cultured human umbilical vein endothelial cells (EC-CM) was used as a source of chemotactic factor, and its chemotactic activity for monocytes was tested by using micropore filter assay in modified Boyden chamber. The effects of tetrandrine, a calcium antagonist, on monocyte migration induced by EC-CM were investigated. The results showed that cultured human umbilical vein endothelial cells secreted a chemotactic factor for monocytes, and the monocyte migration induced by this chemoattractant could be inhibited by tetrandrine at the concentration of 10(-8)-10(-5) mol/L in a dose-dependent manner. Verapamil at the concentration of x10(-8) mol/L also showed the same effect. It is suggested that tetrandrine may inhibit monocyte migration into arterial subendothelial space through antagonism to calcium and thereby suppress atherogenesis.


Increased sensitivity to toluene diisocyanate (TDI) in airways previously exposed to low doses of TDI. Repeated airway exposures to toluene diisocyanate (TDI) may cause sensitization and asthma. This study has examined the acute inflammatory response to TDI in guinea-pig tracheobronchial airways, the development of increased sensitivity to TDI and the effects of xanthines and a glucocorticoid on these responses to TDI. A restricted surface area of the tracheobronchial mucosa of Ketalar-Xylazin anaesthetized guinea-pigs was exposed to TDI, dissolved in olive oil, by means of 1 min infusions through an oral catheter. The TDI-induced inflammatory process was quantified by determination of airway luminal entry of plasma. Already 3 nl (approximately 20 pmol) of TDI produced a significant and sustained exudation response (P less than 0.001 to P less than 0.01, 5 and 17 hr after exposure). Pretreatment with intravenous enprofylline (25 mumol/kg) intraperitoneally or 26 mumol/kg by tracheal superfusion) was without effect. Two repeated exposures to TDI 3 nl (on days 1 and 8) made the animals hyperresponsive to TDI so that on day 15 a previously subthreshold dose of TDI (0.3 nl) produced significant exudation both at 5 and 17 hr after exposure (P less than 0.001 to P less than 0.01). Similarly, two repeated dermal exposures to a large dose of TDI (20 microliters) lowered the threshold for tracheal provocation with TDI. Budesonide (2.6 mumol/kg orally) given daily during the topical airway 'sensitization' regimen (days 1-14) significantly reduced the response to the subsequent 0.3 nl challenge dose of TDI (P less than 0.05). The effects of daily treatments with either theophylline (100 mumol/kg) or enprofylline (50 mumol/kg) were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)


Adhesion, growth, and matrix production by osteoblasts on collagen substrata. A number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel. Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.


Calcium regulating activity of 26,27-dialkyl analogs of 1 alpha,25-dihydroxyvitamin D3. A series of analogs of 1 alpha,25-dihydroxyvitamin D3[1,25(OH)2D3] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3[1,25(OH)2-(Me)2D3], followed by 1,25(OH)2D3, 1 alpha,25-dihydroxy-26,27-diethylvitamin D3[1,25(OH)2(Et)2D3], and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3[1,25(OH)2(Pr)2D3] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)2D3, 1,25(OH)2(Me)2D3, 1,25(OH)2(Et)2D3, and 1,25(OH)2(Pr)2D3 in competing with 1,25(OH)2D3 for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D3 for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)2D3. Effect of 1,25(OH)2(Me)2D3 on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)2D3. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)2D3.


Characterization of gap junctions between osteoblast-like cells in culture. The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4-5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.


Effects of cyclosporin A on rat osteoblasts (ROS 17/2.8 cells) in vitro. The effects of the immunosuppressive drug cyclosporin A (CsA) were evaluated on ROS 17/2.8 cells in vitro. ROS cells were treated with CsA (0, 0.5, 1.0, 5.0 micrograms/ml) for 3 days with and without bovine parathyroid hormone (bPTH) (1-34) 10 nM. CsA at 0.5, 1.0, 5.0 micrograms/ml without PTH and at 5.0 micrograms/ml in the presence of PTH significantly inhibited proliferation, as determined by a tetrazolium colorimetric assay. In addition, ROS cell number was significantly reduced at 3 and 4 days with CsA (5.0 micrograms/ml) without affecting cell viability. Incorporation of [3H]-thymidine into DNA was significantly reduced by 3.0 and 5.0 micrograms/ml CsA after 12 and 24 hours exposure. Basal and 1,25-dihydroxyvitamin D3-stimulated alkaline phosphatase levels in confluent ROS cells were reduced (P less than 0.05) with CsA (1.0 and 3.0 microgramS/ml). Pretreatment of ROS 17/2.8 cells with CsA did not alter PTH-stimulated cAMP levels or [125I]-PTHrP binding to ROS cells. CsA treatment of ROS 17/2.8 cells induced a spindle-shaped appearance with loss of attachment in confluent cultures. When ROS cells were cultured in CsA-containing media, cellular attachment at 6 and 12 hours was reduced (P less than 0.05) compared with untreated ROS cells. These findings indicate that CsA was capable of inhibiting proliferation, cell number, mitogenesis, alkaline phosphatase levels, and cell attachment of ROS cells without affecting PTH binding or cAMP levels. This direct effect of CsA on osteoblasts may be important in changes of bone remodeling observed in CsA-treated humans and animals.


Demonstration of calmodulin-sensitive calcium translocation by isolated osteoclast plasma membrane vesicles. Plasma membrane vesicles were prepared from chicken osteoclasts, and active calcium transport was demonstrated in a spectrofluorimetric assay using the fluorescent calcium concentration indicator, fura-2. Transport activity was inhibited by quercetin (10 microM), sodium vanadate (10 microM), and the anticalmodulin agents, compound 48/80 (20 and 200 micrograms/ml) and calmidazolium (10 and 20 microM). The transport rate (Vmax, 1.3 nmol/mg protein/min) was not altered in the presence of the protonophore, nigericin (1 microM), indicating that proton transport was not driving calcium transport. Release of accumulated calcium in the vesicles occurred with the addition of bromo-A23187 (5 microM) or ionomycin (5 microM). Increasing calcium transport occurred with increasing calcium concentration. Finally, the calmodulin content of the vesicles was demonstrated to be 54-134 U/mg protein. These results demonstrate that a calmodulin-sensitive, ATP-dependent calcium transporter is present in the osteoclast plasma membrane.