Vasodilator effect of scoparone (6,7-dimethoxycoumarin) from a Chinese herb. A possible mechanism of the vasodilator effect of scoparone was investigated. Scoparone (10(-6)-3 x 10(-5) M) dilated rat aortic rings precontracted with phenylephrine in a dose-dependent manner. The presence of endothelium facilitated the vasodilator effect. Scoparone depressed the contractile responses to phenylephrine and serotonin, but not that to potassium chloride. Both the vasoconstriction and O2- production induced by alloxan, a diabetogenic compound, were depressed by scoparone. It appears that scoparone exhibited a free radical scavenger-like effect. The dilatation elicited by acetylcholine was potentiated by scoparone. The dilator activity of scoparone was markedly inhibited by methylene blue and hemoglobin, guanylate cyclase inhibitors. Furthermore, the basal guanosine 3',5'-cyclic monophosphate (cGMP) level was elevated in the presence of scoparone. The dilator activity of scoparone was also inhibited by quinacrine (inhibitor of phospholipase A2) and indomethacin (inhibitor of cyclooxygenase). Our results showed further that the output of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, was enhanced by scoparone. It is suggested that the vasodilator effect of scoparone in rat aorta may be mediated through the enhancement of prostacyclin release, protecting against EDRF inactivation, and activating guanylate cyclase.


PAF antagonism in vitro and in vivo by aglafoline from Aglaia elliptifolia Merr. Aglafoline, isolated from Aglaia elliptifolia Merr, inhibited in a selective and concentration-dependent manner the aggregation and ATP release reaction induced in washed rabbit platelets by PAF (platelet-activating factor). The IC50 values of aglafoline, BN52021 and kadsurenone on PAF (3.6 nM)-induced platelet aggregation were about 50, 12 and 18 microM, respectively. Aglafoline also inhibited [3H]PAF (3.6 nM) binding to washed rabbit platelets with an IC50 value of 17.8 +/- 2.6 microM. The concentration-response curve of PAF-induced platelet aggregation was shifted to the right by aglafoline with pA2 and pA10 values of 5.97 and 5.04, respectively. Although thromboxane B2 formation caused by collagen and thrombin was partially suppressed by aglafoline, thromboxane B2 formation caused by ionophore A23187 and arachidonic acid was not affected. Aglafoline inhibited the [3H]inositol monophosphate formation caused by PAF but not that caused by collagen or thrombin in the presence of indomethacin (20 microM). The cAMP content of washed rabbit platelets was not affected by aglafoline. Rat femoral intravenous administration of aglafoline (10 mg/kg) did not affect blood pressure. However, aglafoline (10 mg/kg) both prophylactically and therapeutically antagonized PAF (2.5 micrograms/kg)-induced hypotensive shock in rats. Intravenous PAF (30 ng/kg) caused severe bronchoconstriction in guinea pigs. This effect was completely blocked by aglafoline. This implies aglafoline is an effective PAF antagonist not only in vitro, but also in vivo.


Increased acetylcholine and quisqualate responsiveness after blockade of GABAB receptors. The aim of this study was to gain further insight into the function of cortical GABAB receptors. In chloral hydrate-anaesthetized rats, microiontophoretic administration of the GABAB receptor blocker CGP 35348 induced a moderate increase in firing of spontaneously active neurons in the rostral and caudal sensorimotor cortex. This increase in cell firing was accompanied by a reduction in the baclofen-induced inhibition of cell activity. In contrast to the GABAA receptor antagonist bicuculline methiodide, CGP 35348 did not induce any paroxysmal discharges. The excitatory responses of rostral cortical neurons elicited by iontophoretically applied acetylcholine and quisqualate were potentiated in most neurons after both microiontophoretic and intravenous administration of CGP 35348. The potentiation was observed in the absence of any change in the spontaneous firing rate. These effects were dose-dependent for both routes of administration. The potentiation of the quisqualate response was reversed by intravenously applied baclofen. In conclusion, these findings suggest that cortical GABAB receptors are involved in the control of cortical neuronal excitability.


Antinociceptive effect of L-arginine on the carrageenin-induced hyperalgesia of the rat: possible involvement of central opioidergic systems. L-arginine is considered to be a precursor substance of kyotorphin (tyrosyl-arginine), a [Met5]enkephalin releaser with antinociceptive action. We examined the antinociceptive effect of L-arginine in rats. L-Arginine (300-1000 mg/kg) administered subcutaneously (s.c.) elicited antinociception (assessed by the Randall-Selitto method) in rats with a carrageenin-treated hindpaw. Naloxone (2 mg/kg s.c.) but not N-methyl-levallorphan (20 mg/kg s.c.), a peripherally selective opioid antagonist, inhibited L-arginine-induced antinociception. Intracerebroventricular administration of L-arginine (0.2-1.0 mg/rat) produced a dose-related inhibition of the carrageenin-induced hyperalgesia. Intraplantar (i.pl.) injection of L-arginine (0.5-1.0 mg/paw) also induced antinociception, which was resistant to naloxone (2 mg/kg s.c.) but was antagonized by methylene blue (0.5 mg/paw i.pl.), a guanylate cyclase inhibitor. L-Arginine (1000 mg/kg s.c.) did not inhibit edema formation in the carrageenin-treated rat hindpaw. These results suggest that systemically administered L-arginine produces mainly an antinociceptive effect mediated by central opioidergic mechanisms in rats with carrageenin-induced hyperalgesia.


Evidence that an atypical beta-adrenoceptor mediates the prejunctional inhibition of non-adrenergic non-cholinergic contraction in guinea-pig bronchi. We investigated the effect of the putative beta 3 agonist BRL 35135 on non-adrenergic non-cholinergic (NANC) contractions in guinea-pig bronchial strips. BRL 35135 (10(-9) to 10(-6) M) did not alter the baseline tension but reduced NANC contractions induced by electrical field stimulation (EFS) in a concentration-dependent fashion without having a significant effect on the contraction induced by substance P (10(-6) M). BRL 35135 (10(-6) M) also reduced the contraction induced by capsaicin (10(-7) M). Likewise, BRL 37344 (10(-9) to 10(-6) M) reduced NANC contractions induced by EFS in a concentration-dependent fashion. While BRL 37344 up to concentrations of 10(-8) M did not alter the contraction induced by SP (10(-6) M), BRL 37344 (10(-8) M) significantly inhibited NANC contractions induced by EFS and capsaicin (10(-7) M), (P less than 0.01). The inhibitory effect of BRL 35135 (10(-6) M) on NANC contractions induced by EFS was not significantly altered by the non-selective beta-adrenoceptor antagonists, propranolol and pindolol (P greater than 0.10), by the beta 1-selective antagonists, atenolol and metoprolol (P greater than 0.20) (10(-8) to 10(-6) M), or by the alpha-adrenoceptor antagonist, phentolamine (10(-7) to 10(-5) M) (P greater than 0.50). These results suggest that beta 3 agonists exert a prejunctional inhibitory action on NANC contractions.


A highly selective delta 1-opioid receptor antagonist: 7-benzylidenenaltrexone. In guinea pig brain membranes 7-benzylidenenaltrexone (BNTX) possesses 100-fold greater affinity (Ki = 0.1 nM) for [3H]DPDPE [3H][D-Pen2,D-Pen5]enkephalin) binding sites (delta 1) relative to those of [3H]DSLET ([3H][D-Ser2,Leu5]enkephalin-Thr6) (delta 2). The ED50 dose ratio (tail flick) in mice for the antagonism of DPDPE-induced antinociception of BNTX (6.3 pmol i.c.v.) was 7.2, whereas for DSLET, morphine and U69593 it was not significantly different from unity. The fact that there was no correlation of the binding or in vivo data for BNTX with antagonist potency in smooth muscle preparations suggests that the in vitro pharmacologic activity is mediated by delta-opioid subtypes that are different from those in the brain.


Vasoactive intestinal peptide and helodermin inhibit the release of cyclo-oxygenase products induced by leukotriene D4 and bradykinin from guinea-pig perfused lung. Vasoactive intestinal peptide (VIP, 10 nM) inhibited the release of cyclo-oxygenase products, detected by both bioassay and radioimmunoassay, induced by leukotriene (LT) D4 (3-30 pmol) and bradykinin (BK, 3-30 nmol) from guinea-pig isolated perfused lung. Helodermin (10 nM), a peptide that is structurally related to VIP, and salbutamol (10 nM), a beta 2-adrenoceptor agonist, evoked a similar inhibitory effect on LTD4-induced release of cyclo-oxygenase products. The generation of TxB2 and 6-keto-PGF1 alpha following stimulation with exogenously administered arachidonic acid (30-300 nmol) was not significantly attenuated in the presence of either VIP, helodermin or salbutamol. These results show that VIP, helodermin and salbutamol are potent inhibitors of the release of cyclo-oxygenase products induced by agonists known to activate endogenous arachidonic acid metabolism in guinea-pig lung. Since the metabolism of exogenously administered arachidonic acid was not inhibited these results suggest that the inhibitory effect may be exerted on events preceding the mobilisation of arachidonic acid and may involve cyclic AMP.


Increased density of alpha-adrenoceptors in vas deferens of spontaneously hypertensive rats (SHR), indicated by functional and receptor binding studies. Pharmacological parameters were determined from contractile responses mediated by alpha-adrenoceptors in vas deferens from spontaneously hypertensive rats (SHR) and corresponding normotensive controls, Wistar Kyoto rats (WKY), and compared with data obtained from radioligand binding assays. Contractile responses induced in longitudinal and circular muscle layers by the alpha-adrenoceptor agonist noradrenaline (NA) and by barium chloride were recorded as described previously. In both muscle layers the maximal effects induced by NA, but not by BaCl2, were significantly greater in SHR. As a consequence, the relative responsiveness ratio (rho) for the alpha-adrenoceptor was also larger for SHR than for WKY. NA-induced contractions of both muscle layers were competitively antagonized by indoramine. The pA2 values for indoramine and pD2 values for NA were the same in SHR and WKY, indicating that alpha-adrenoceptor affinity was not changed in SHR. Additionally, binding studies with the alpha-adrenoceptor ligand [3H]WB4101 revealed that Bmax values were greater in the vas deferens of SHR, whereas Kd values were not significantly different from those of WKY controls. In summary, although differences could not be detected for affinity-related parameters, a greater density of alpha-adrenoceptors was shown for SHR in receptor binding studies and this was corroborated by functional studies.


Antidiarrheal and colonic antipropulsive effects of spinal and supraspinal administration of the natural delta opioid receptor agonist, [D-Ala2]deltorphin II, in the rat. The ability of the natural selective delta opioid receptor agonist, [D-Ala2]deltorphin II (DADELT II), to inhibit the diarrhea induced by castor oil and colonic glass bead expulsion, was studied in rats after supraspinal and spinal administration. When injected intracerebroventricularly, DADELT II (0.2, 1, 10 micrograms/rat) inhibited diarrhea and colonic bead expulsion in a dose-related fashion but did not affect the rate of small intestine transit. Similar results were obtained when the same dose of DADELT II was administered spinally. The antidiarrheal and colonic antipropulsive effects of supraspinally and spinally DADELT II were partially or completely antagonized by subcutaneous pretreatment with 1 and 10 mg/kg, respectively, of naltrindole, a selective delta opioid receptor antagonist. These findings indicate that, in the rat, supraspinal and spinal delta opioid receptors play a role in modulating diarrhea and colonic propulsion and that DADELT II is a useful tool for investigating the role of the delta opioid system in gastrointestinal function.


Influence of omega-conotoxin on morphine analgesia and withdrawal syndrome in rats. The effect of omega-conotoxin on opiate analgesia and withdrawal syndrome was investigated in rats. omega-Conotoxin given i.c.v. and i.p. caused weak analgesia in the tail-flick test. When the toxin (20 ng/rat) was given i.c.v. immediately before morphine (1.5 micrograms/rat i.c.v.) the resultant analgesic effect was additive. In contrast, the analgesia elicited by morphine (3 micrograms/rat i.c.v.) was greatly reduced after 24-h pretreatment with the toxin (20 ng/rat i.c.v.). The systemic administration of the toxin (10 micrograms/kg i.p.) did not affect morphine analgesia whether omega-conotoxin was coadministered with morphine (2.5 mg/kg i.p.) or was given 24 h before the opiate (5 mg/kg i.p.). omega-Conotoxin i.c.v. injected in morphine-dependent rats 15 min before naloxone challenge significantly attenuated the abstinence syndrome. On the contrary systemic administration of omega-conotoxin failed to suppress the morphine withdrawal syndrome. The present results suggest that omega-conotoxin affects both acute and chronic effects of morphine.


Taurine inhibition of fast Na+ current in embryonic chick ventricular myocytes. Effects of taurine on the fast Na+ current (INa) in 17-day-old embryonic chick ventricular myocytes were examined using the whole-cell voltage-clamp technique. The cells were spherical (10-15 microns diameter) and had a capacitance of 9.8 +/- 1.3 pF. The experiments were performed at room temperature (22 degrees C), and the holding potential was -90 mV. After the patch membrane was broken, peak INa initially increased, and then decreased and became stable within 3-5 min. The experiments on taurine were started after INa had stabilized. The characteristics of INa were as expected, including sensitivity to tetrodotoxin (10 microM). When added to the bath, taurine inhibited INa and shifted the reversal potential in the hyperpolarizing direction. At 10 mM, taurine inhibited INa by 38.2 +/- 4.3%, and shifted the reversal potential by 10.2 +/- 3.1 mV. The time to peak current was slowed: 0.83 +/- 0.20 ms (n = 11) in control, 1.03 +/- 0.18 ms (n = 9) in 10 mM taurine, and 1.10 +/- 0.19 ms (n = 10) in 20 mM taurine. These effects of taurine were not reversed by 30 min washout. At low concentrations, taurine actually enhanced INa in 3 of 8 cells at 1 mM, and in 4 of 10 cells at 5 mM; the reversal potential was still shifted in the hyperpolarizing direction by 5.7 +/- 1.6 mV. The time course of inactivation (fitted as a single exponential at test potential of -30 mV) was not affected: 1.1 +/- 0.5 ms in control 1.2 +/- 0.4 ms at 10 mM taurine.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification and initial characterization of a putative neuromedin B-type receptor from rat urinary bladder membranes. Receptor binding site(s) on the rat urinary bladder membranes were characterized using a biologically active analog of bombesin, [Tyr4,Leu14]bombesin, and a 50,000 x g total particulate preparation. The binding was specific, reversible, saturable, time- and concentration-dependent. A dissociation curve showed that both bombesin and neuromedin B equally displaced the radioligand in the first 10 min after saturation. From the rate constant of association K + 1 = 7.60 x 10(9) M-1 min-1, and the rate constant of dissociation k-1 = 0.050 min-1, the apparent equilibrium dissociation constant Kd = 6.57 +/- 1.09 pM was determined. A linear Scatchard plot of the specific binding of 125I-[Tyr4,Leu14]bombesin to the membranes revealed that the radioligand bound with high affinity, Kd = 6.38 +/- 0.86 pM, to a single class of sites (Bmax = 2.3 fmol/mg protein). The Hill coefficient of the same binding data was 1.05 +/- 0.21, indicating that the radioligand was binding to a single population of noninteracting binding sites. Both bombesin and neuromedin B displaced the radioligand dose dependently (IC50 = 0.3 nM). Neurokinin A and neurokinin B were less potent (IC50 = 20 and 110 nM, respectively). Substance P, or the specific bombesin receptor antagonists [D-Phe6]bombesin-(6-13) methyl ester, [D-F5Phe6,D-Ala11]bombesin-(6-11) methyl ester, [D-Phe6]bombesin-(6-13) propylamide, [D-Phe6,Leu13psi(CH2NH)Leu14]bombesin or [D-Cpa6,Phe14(psi13-14)]bombesin-(6-14) had an IC50 greater than 1 microM. The results presented suggest the presence of neuromedin B receptor sites on the rat urinary bladder membranes that can be occupied also by some other peptides, notably bombesin, neurokinin A and neurokinin B.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiac electrical responses to catecholamines are differentially mediated by beta 2-adrenoceptors in anesthetized dogs. We investigated the beta 2-adrenoceptor-mediated effects of atrial and ventricular effective refractory period (ERP), SA node pacemaker activity, and AV conductivity induced by sympathetic nerve stimulation or epinephrine infusion in anesthetized dogs. A beta 2-adrenoceptor antagonist, ICI 118,551 up to 100 micrograms/kg, i.v., inhibited the positive chronotropic and dromotropic responses to sympathetic stimulation but did not shorten the atrial or ventricular ERP, ICI 118,551 also attenuated the positive chronotropic and dromotropic responses and the shortening of atrial ERP in response to epinephrine but not the shortening of ventricular ERP. A selective beta 1-adrenoceptor antagonist, atenolol, inhibited each electrical cardiac response to sympathetic stimulation and epinephrine infusion in a similar manner. These results suggest that beta 2-adrenoceptor-mediated electrical cardiac responses to endogenous catecholamines also exist in addition to the predominant beta 1-adrenoceptor-mediated responses, and that the order of the proportion of beta 2-adrenoceptor-mediated cardiac effects was SA node pacemaker activity much greater than AV conductivity = atrial ERP much greater than ventricular ERP in the dog heart.


A novel treatment of global cerebral ischaemia with a glycine partial agonist. Chronic treatment of gerbils with 1-aminocyclopropanecarboxylic acid (a high affinity, partial agonist at strychnine-insensitive glycine receptors) resulted in a 3-fold increase in survival, a significant improvement in neurological status, and an extensive protection of vulnerable brain regions following severe forebrain ischaemia. A bolus of 1-aminocyclopropanecarboxylic acid 30 min prior to ischaemia did not further improve outcome compared to gerbils receiving their last injection 24 h prior to ischaemia. These findings are consistent with the hypothesis that chronic treatment with a glycine partial agonist desensitizes the N-methyl-D-aspartate receptor complex. Pharmacological intervention at the strychnine-insensitive glycine receptor may be an effective means of ameliorating the consequences of neuronal degeneration caused by excitotoxic phenomena.


Vasorelaxation of rat thoracic aorta caused by osthole isolated from Angelica pubescens. The pharmacological effects of osthole on isolated rat thoracic aorta were examined. Osthole inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta in a concentration-dependent manner (40-200 microM). The tonic contraction elicited by NE was also relaxed by the addition of osthole. This relaxing effect of osthole was not affected by indomethacin (20 microM) and was still observed in endothelium-denuded rat aorta. Methylene blue (50 microM) partially antagonized this relaxing effect of osthole. In high-K+ medium (80 mM), the Ca2+ (0.03-3 mM)-induced vasocontraction was inhibited concentration dependently by osthole (20-100 microM). Addition of osthole (100 microM) at the plateau of the K+ (80 mM)-induced contraction caused relaxation. Methylene blue (50 microM) did not antagonize this relaxation. In Ca(2+)-free medium, the caffeine (10 mM)-induced phasic contraction was also suppressed by osthole in a concentration-dependent manner. Although the cAMP level was not changed by osthole, the cGMP level of rat aorta was increased by osthole in a concentration-dependent manner. The increase in cGMP level caused by osthole was completely blocked by methylene blue. [3H]Inositol monophosphate formation caused by NE was not affected by osthole at a concentration of 200 microM. The 45Ca2+ influx elicited by either NE or high K+ was inhibited by osthole in a concentration-dependent manner. It is concluded that osthole relaxes rat thoracic aorta by virtue of its Ca(2+)-channel blocking properties and by elevating cGMP levels in vascular smooth muscle.


Release of corticotropin-releasing factor from superfused rat hypothalami induced by interleukin-1 is not dependent on adrenergic mechanism. Interleukin-1 (IL-1) is a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis. The hypothalamus seems to be the most important site of action of IL-1 on the HPA axis, inducing corticotropin-releasing factor (CRF) secretion. Catecholamines are important modulators of CRF secretion. In turn, IL-1 stimulates catecholamine release from the hypothalamus. In the present study, we examined the possible involvement of hypothalamic catecholamines in the effect of IL-1 beta on hypothalamic CRF secretion, by using an in vitro rat hypothalami continuous perifusion system. Neither in vivo pretreatment with an inhibitor of catecholamine synthesis nor in vitro exposure to alpha- or beta-adrenoceptor antagonists (phenoxybenzamine or propranolol, respectively) nor combination of both treatments altered the effect of IL-1 on CRF secretion from superfused hypothalami. These data indicate that catecholamines are not involved in the in vitro stimulatory action of IL-1 on hypothalamic CRF secretion.


Contribution of antiplatelet activity to the effects of 5-HT2 receptor antagonists on reperfusion-induced arrhythmias in anaesthetized rats. The effects of certain 5-HT receptor antagonists were examined on ischaemia-induced and reperfusion-induced arrhythmias, on ex vivo platelet aggregation and on isolated cardiac muscle. Methiothepin (1 mg kg-1) reduced the total number of ischaemia-induced ventricular premature beats whereas ICI 170,809 (1 mg kg-1) reduced reperfusion-induced mortality to 10% compared with 70% in controls. ICI 169,369 did not significantly alter either ischaemia- or reperfusion-induced arrhythmias. High concentrations of both ICI 169,369 and ICI 170,809 caused reductions in the maximum driving frequency of isolated cardiac muscle but methiothepin had no significant effect. Administration of ketanserin, ritanserin, methiothepin or ICI 170,809, but not ICI 169,369, abolished the ability of 5-HT to enhance platelet aggregation. The results of these experiments suggest that the ability of 5-HT2 receptor antagonists to reduce reperfusion-induced arrhythmias may be related to their antiplatelet activity.


Kappa 3 opiate receptor binding in the mouse and rat. Previous work examining the binding of [3H]naloxone benzoylhydrazone (NalBzoH) in calf brain has identified a novel binding site, kappa 3. In mice and rats NalBzoH elicits an analgesic response which can clearly be differentiated from classical mu, delta or kappa 1 mechanisms and which is pharmacologically consistent with a kappa 3 receptor mechanism of action. In the current studies we demonstrate the presence of kappa 3 sites in both mouse and rat brains. The selectivity of the kappa 3 sites for opioids clearly discriminated it from traditional mu, kappa 1, kappa 2 or delta receptors. In the rat, the density of kappa 3 sites increased 2.5-fold from age 2 days to 21 days, after which it remained relatively stable. Among a number of brain regions in the rat, the density of kappa 3 sites varied dramatically. Highest levels were present in the hypothalamus, thalamus, striatum and midbrain with very low levels in the cerebellum. Intermediate levels were present in cortex, brain stem and spinal cord. Together, these studies support the presence of kappa 3 receptors in both mouse and rat brain which are very similar to those previously reported in calf brain.


GABAA receptor subunit mRNA levels are differentially influenced by chronic FG 7142 and diazepam exposure. Levels of mRNA for the alpha 1, gamma 2 and beta 1 subunits of the GABAA receptor complex were examined in rats maintained on a chronic, continuous schedule of exposure to the benzodiazepine inverse agonist FG 7142. The effect of chronic exposure to the benzodiazepine agonist diazepam was also examined on levels of gamma 2 subunit mRNA. FG 7142 (2 mg/ml of 100% dimethyl sulfoxide (DMSO) or vehicle (100% DMSO) was administered continuously for 8 days in the right ventricle via an osmotic minipump. At the end of the eighth day of exposure, the brain was removed and cerebral cortex, cerebellum and hippocampus were dissected and mRNA prepared from each region. Levels of GABAA alpha 1 and gamma 2 subunit mRNA were examined by Northern blot analysis with cDNA probes specific for these subunits. A significant increase in alpha 1 mRNA was measured in both cortex and hippocampus, but not in cerebellum, of rats chronically exposed to FG 7142 relative to vehicle-treated rats. A significant increase in gamma 2 subunit mRNA in cortex was also evident in drug-treated rats; however, no change in gamma 2 subunit mRNA was observed in either the hippocampus or cerebellum. Examination of GABAA beta 1 subunit mRNA by solution hybridization using a beta 1 riboprobe revealed no effect of chronic FG 7142 treatment on this subunit in either cortex, hippocampus or cerebellum. In rats chronically exposed to diazepam (21 days via silastic implants), levels of gamma 2 subunit mRNA were significantly decreased in cortex, but not changed in either hippocampus or cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)


The alpha 1-adrenoceptor is inactivated by alterations in membrane phospholipids. The influence of the membrane environment on the alpha 1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the alpha 1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic alpha 1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the alpha 1-adrenoceptor.


Pharmacological studies on CCKB receptors in guinea pig synaptoneurosomes. Preliminary studies on CCK receptors in the central nervous system were carried out on guinea pig cerebral cortical synaptoneurosome preparations. In binding assays, the range of affinity of CCK-8, Boc-[Nle28,Nle31]CCK-7, a potent CCK analog, Boc-[Leu31]CCK-4 and of the two benzodiazepine CCK receptor antagonists L-365,260 and MK-329, is in agreement with the presence of CCKB receptors on this model. The effects of Boc-[Nle28,Nle31]CCK-7 on inositol phosphates, cAMP accumulation and 45Ca2+ efflux were investigated. Neither inositol phosphate nor cAMP accumulations could be observed. On the other hand, evidence of Boc-[Nle28,Nle31]CCK-7-, CCK-8- and Boc-[Leu31]CCK-4-induced 45Ca2+ efflux was found in a dose-dependent manner. The CCKB-selective receptor antagonist L-365,260 and, with a weaker efficiency, the CCKA-selective receptor antagonist MK-329, are able to block a maximal effect of Boc-[Nle28,Nle31]CCK-7-induced 45Ca2+ efflux, suggesting that CCKB receptors may regulate calcium ion mobilization.


Effects of chronic anethole trithione and amitriptyline treatment on rat parotid gland signalling. The present study examines the mechanism(s) of action of anethole trithione (Sulfarlem S25) compared to the sialogogue pilocarpine. The chronic effects (7 days of treatment) of anethole trithione, pilocarpine and/or amitriptyline on autonomic receptor binding (homogenates) were measured together with parallel tests of stimulation-induced rises in delta [Ca2+]i in collagenase-isolated rat parotid acini. The results revealed that chronic treatment with amitriptyline resulted in significantly increased rises in delta [Ca2+]i after stimulation with 20 microM of carbachol or adrenaline, and a significant increase in muscarinic acetylcholine receptor density. In addition, anethole trithione also increased cholinergic and adrenergic responsiveness. The double treatment of amitriptyline and anethole trithione or amitriptyline and pilocarpine did, however, prevent the rise in delta [Ca2+]i observed under conditions when these drugs were administered alone. Furthermore, anethole trithione, but not pilocarpine, was able to prevent the amitriptyline-induced upregulation in muscarinic acetylcholine receptor density. In conclusion, the experimental data presented in this study are compatible with the hypothesis that anethole trithione might stimulate some post-receptor effect in the coupling to the secretory response. In addition, this study supports the beneficial effects of anethole trithione in treating drug-induced xerostomia.


Expression of Fos-like immunoreactivity by yohimbine and clonidine in the rat brain. To elucidate the role of alpha 2-adrenoceptors in transcriptional control in the rat brain, we localized the Fos-like immunoreactivity (Fos-LI) induced by alpha 2-adrenoceptor agonists and by an antagonist. Injections of yohimbine (5 mg/kg, i.p.) into rats led to the induction of Fos-LI in areas with a dense alpha 2-adrenoceptor binding such as the locus coeruleus, the bed nucleus of stria terminalis, the central nucleus of amygdaloid complex, the paraventricular nucleus, the nucleus tractus solitarius, and ventrolateral medulla oblongata. Clonidine (500 micrograms/kg, i.p.) suppressed the Fos expression by yohimbine in these nuclei, and clonidine (100 micrograms/kg, i.p.) or guanabenz (4 mg/kg, i.p.) induced Fos-LI in oxytocin neurons in the paraventricular and supraoptic nuclei in the hypothalamus. Thus, the alpha 2-adrenoceptor is involved in transcriptional control via Fos expression in neurons related to autonomic and other functions.


Selective dopaminergic mechanism of dopamine and SKF38393 stimulation of inositol phosphate formation in rat brain. We have previously reported that dopamine and the D1 receptor-selective agonist, SKF38393, stimulate the formation of inositol phosphates in rat brain slices (Undie and Friedman, 1990, J. Pharmacol. Exp. Ther. 253, 987). The present experiments were conducted to determine if actions at alpha-adrenoceptors or at serotonergic sites may contribute to, or interact with, the observed stimulation of phosphoinositide hydrolysis by dopamine receptor agonists. Rat striatal slices prelabeled with [3H]inositol were treated with up to 500 microM dopamine, norepinephrine, serotonin (5-HT), or the dopamine D1 receptor agonist, SKF38393, and accumulated inositol phosphates determined. The action of norepinephrine was dose-dependently blocked by the selective alpha 1-adrenoceptor antagonist, prazosin, but not by SCH23390. The actions of dopamine and SKF38393 were dose-dependently blocked by the dopamine D1 receptor antagonist, SCH23390, but not by prazosin. The effects of 5-HT were blocked by the nonselective 5-HT antagonist, methiotepin, the selective 5-HT2 antagonist, ketanserin, the mixed 5-HT2/5-HT1C antagonist, mianserin, and, with much less potency, by the selective 5-HT1C antagonist, mesulergine. On the contrary, the serotonin receptor antagonists did not block the response to SKF38393, and there was no dose-dependent blockade of the 5-HT response by SCH23390. These observations indicate that the actions of dopamine and SKF38393 in stimulating inositol phosphate formation are selectively mediated through a D1-like dopamine receptor.


Effect of electroconvulsive shock on 5-HT2 and alpha 1-adrenoceptors and phosphoinositide signalling system in rat brain. We studied the effect of repeated administration of electroconvulsive shock (ECS) on alpha 1-adrenoceptor subtype (alpha 1A and alpha 1B) and 5-HT2 (serotonin-2) receptors and receptor-mediated phosphoinositide (PI) hydrolysis in rat cerebral cortex. We observed that repeated administration with ECS significantly increased the density of 5-HT2 receptors, as labeled by [3H]ketanserin, as well as 5-HT-stimulated [3H]inositol-1-phosphate ([3H]IP1) in rat cerebral cortex. We also observed that repeated ECS administration caused a significant increase in the number of alpha 1-adrenoceptors and the alpha 1B-adrenoceptor subtype as measured by (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone binding. However, it had no significant effects on norepinephrine (NE)-stimulated [3H]IP1 formation or alpha 1A-adrenoceptor subtype. These results thus suggest that up-regulation of 5-HT2 receptors after administration with ECS is associated with increased 5-HT-stimulated [3H]IP1 formation. The lack of effects on NE-stimulated PI turnover in ECS treated rats may be due to its lack of effect on the alpha 1A-adrenoceptor subtype.


Bi-directional changes in the levels of messenger RNAs encoding gamma-aminobutyric acidA receptor alpha subunits after flurazepam treatment. Changes in gamma-aminobutyric acidA (GABAA) receptor function have been observed following chronic benzodiazepine administration. The molecular mechanisms responsible are unknown, but one possibility is that benzodiazepines induce alterations in the expression of genes which encode subunits of the GABAA receptor complex, resulting in changes in the receptor structure and function. We have investigated this hypothesis by evaluating the effect of flurazepam 40 mg/kg i.p. on brain levels of the mRNAs which encode the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA receptor complex. Rats were treated with flurazepam or vehicle for up to 32 days. No changes were found in the levels of alpha 1 and alpha 2 mRNA. A rapid decrease was found in the level of alpha 5 mRNA; alpha 3 mRNA was increased by 4 days of treatment and this was followed by an increase in alpha 6 levels. These results support the hypothesis that the alteration in GABAA receptor function after benzodiazepine administration results from changes in subunit gene expression. Furthermore, the predicted consequences of the pattern of mRNA changes we have observed suggest that altered gene expression may be important in the genesis of benzodiazepine tolerance.


Carbachol stimulates adenylate cyclase and phospholipase C and muscle contraction-relaxation in a reciprocal manner in dog iris sphincter smooth muscle. In the dog iris sphincter, muscarinic acetylcholine receptors are coupled either to the stimulation of phospholipase C and muscle contraction or to the stimulation of adenylate cyclase and muscle relaxation, this was found to be dependent upon the concentration of the muscarinic agonist. In contrast to the dog, muscarinic receptors in iris sphincters from different mammalian species were found to be coupled to phospholipase C and contraction at all concentrations of carbachol investigated (1-100 microM). In the dog sphincter, lower concentrations (less than 5 microM) of carbachol stimulated myo-inositol 1,4,5-trisphosphate (IP3) production, inhibited cAMP formation and induced contraction, and higher concentrations (greater than 5 microM) enhanced cAMP formation, inhibited IP3 production and induced relaxation. The mechanisms for the stimulatory effects on cAMP formation through muscarinic receptors were investigated. Carbachol (25 microM) increased both basal and isoproterenol- and forskolin-stimulated cAMP levels. Atropine inhibited the carbachol-stimulated increase in cAMP levels in a dose-dependent manner with an IC50 of 9 nM. Intracellular Ca2+, derived from IP3-induced Ca2+ release and/or from muscarinic receptor-operated Ca2+ influx, and protein kinase C may mediate the muscarinic receptor-linked rise in intracellular cAMP. This conclusion is supported by the following findings. (1) At short time intervals (less than 1 min) carbachol (25 microM) increased IP3 production and contraction and this was followed (between 1 and 20 min) by cAMP formation and muscle relaxation. (2) Carbachol-stimulated IP3 production was detected at a concentration of the agonist 26-fold lower than that required for cAMP formation, and it was completely blocked by the phorbol ester, phorbol 12,13-dibutyrate (50 nM). (3) A Ca(2+)-calmodulin stimulated adenylate cyclase was demonstrated in membranes from dog iris sphincter but not in that from rabbit and bovine. (4) Trifluoperazine (0.1 microM), a calmodulin antagonist, inhibited the carbachol-stimulated cAMP accumulation. (5) The Ca2+ ionophore A23187 and the phorbol ester increased cAMP production in a dose-dependent manner. A23187 potentiated cAMP production induced by either carbachol or by the phorbol ester. (6) Muscarinic stimulation of cAMP production persisted even after the tissue was pretreated with the phorbol ester or staurosporine. (7) Nifedipine (0.01-0.5 microM), a Ca2+ channel antagonist, inhibited carbachol stimulation of cAMP production, suggesting the presence of a muscarinic receptor-operated Ca2+ influx pathway in this tissue.(ABSTRACT TRUNCATED AT 400 WORDS)


Northern blot and ribonuclease protection study of alpha 2-adrenoceptor subtypes in cultured cell lines. Northern blot and ribonuclease protection assay were used to identify alpha 2-adrenoceptor subtypes in human colonic adenocarcinoma (HT29), neuroblastoma x glioma rat-mouse hybrid NG108-15 (NG108) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the alpha 2-adrenoceptor expressed in HT29, NG108 and OK cells represent the pharmacological alpha 2A, alpha 2B and alpha 2C subtypes respectively. In our Northern blot analysis, hybridization of poly(A)+ RNA from HT29, NG108 and OK cells with human kidney alpha 2-adrenoceptor cDNA probe (alpha 2-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. Hybridization with a human platelet alpha 2-adrenoceptor genomic probe (alpha 2-C10) resulted in two bands for HT29 cells with the size of 4.4 kb and 3.9 kb. No bands were seen for HT29, NG108 and OK cells when hybridized with a third alpha 2-adrenoceptor human genomic DNA probe which is localized in chromosome 2 (alpha 2-C2). For the HT29 cells, the 3.9 kb band was seen only when using the alpha 2-C10 probe. Thus, this band probably represents alpha 2-C10 mRNA. To further characterize the alpha 2-adrenoceptor mRNA expressed in HT29, NG108 and OK cells, the sensitive ribonuclease protection assay was performed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an alpha 2-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with alpha 2-C10 RNA probe and digestion with RNAases protected a 500 bp fragment.(ABSTRACT TRUNCATED AT 250 WORDS)


ACTH binds to [3H]MK-801-labelled rat hippocampal NMDA receptors. The molecular mechanism of the clinical antiepileptic/antimyoclonic action of adrenocorticotrophic hormone (ACTH) is unknown. To explore the possible role of excitatory amino acid receptors, we studied the influence of ACTH and ACTH fragments in vitro on the binding of [3H]MK-801 to rat hippocampus, a region relevant to epilepsy. ACTH-(1-39), ACTH-(1-24), and ACTH-(1-17) displayed micromolar affinities compared to the nanomolar affinity of MK-801, whereas ACTH-(4-10) and four clinically used anticonvulsants were inactive. These findings are not specific for NMDA receptors but conform to the rank order of potency of ACTH fragments at other receptor binding sites.


Theophylline inhibits late asthmatic reactions induced by toluene diisocyanate in sensitised subjects. Toluene diisocyanate (TDI)-induced asthma is a frequent occupational airway disease. To determine whether a calibrated dosage of oral slow-release theophylline inhibits asthmatic reactions and the associated increase of airway responsiveness to methacholine induced by TDI, we examined six asthmatic subjects who developed a late or a dual asthmatic reaction after TDI inhalation challenge. We administered oral slow-release theophylline or placebo to each subject for 7 days according to a double-blind, randomized, cross-over study design. When the subjects received a placebo, TDI caused a late or a dual asthmatic reaction. When the subjects received theophylline. TDI caused significantly reduced late asthmatic reactions. Mean serum theophylline concentrations were within the therapeutic range. Theophylline neither modified the baseline airway responsiveness to methacholine, nor the increase of airway responsiveness to methacholine induced by TDI. These results suggest that slow-release theophylline may improve TDI-induced late asthmatic reactions, but it does not change the baseline airway responsiveness to methacholine and the increase of airway responsiveness to methacholine induced by TDI.


Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid cell lines. The clinical phenotype of Werner's syndrome (WS) includes short stature, premature cataracts, skin atrophy, osteoporosis, graying and loss of hair, neoplasia, diabetes mellitus, and arteriosclerosis. Cultured cells from patients with this autosomal recessive disorder exhibit chromosomal instability and a markedly reduced replicative lifespan and growth rate. To elucidate the cell cycle alterations associated with the growth deficit, we continuously labeled lymphoid cell lines from five WS patients and from four healthy adult controls with 5-bromodeoxyuridine. Bivariate Hoechst 33258/ethidium bromide flow cytometry revealed a 2.4-h prolongation in the minimal duration of the S phase of WS cells (P less than 0.005). Moreover, the fraction of proliferating cells irreversibly arrested in the S phase (5.4% vs 1.4% in controls) was significantly elevated in WS (P less than 0.001). Other cell cycle compartments were not significantly affected in WS cell lines. As a partial test of the hypothesis that the WS phenotype is due to a defect in DNA topoisomerase I (topo I) or DNA topoisomerase II (topo II) we exposed lymphoid cells from a healthy control to the topo I inhibitor camptothecin or to the topo II inhibitor 4'-(9-acridinylamino)methanesulfon-m-anisidine. The cell kinetic alterations elicited by these compounds differed from that exhibited by untreated WS patients. Thus, a primary defect in topo I or II is unlikely in WS. Our cell cycle results, however, provide important evidence that the biochemical genetic lesion is in fact expressed in lymphoblastoid cell lines, the most readily available cells from such subjects.


Murine melanoma cell differentiation and melanogenesis induced by poly(ADP-ribose) polymerase inhibitors. Murine melanoma cells treated with the melanocyte-stimulating hormone (MSH) family of peptides undergo differentiation characterized by enhanced melanogenesis and altered morphology. These effects are mediated via the adenylate cyclase-cAMP pathway leading to activation of protein kinase A (PKA). We have discovered that inhibition of a post-translational modification of chromatin proteins, viz. poly(ADP-ribosylation), also induces melanogenesis and differentiation in these cells. A range of competitive inhibitors (benzamide and its derivatives) of the nuclear enzyme poly(ADP-ribose) polymerase (PADPRP; EC 2.4.2.30) was utilized, and their ability to induce melanogenesis reflected their potency as PADPRP inhibitors. These compounds induced melanogenesis at low doses (20 microM-2 mM) which did not affect cell growth or viability. Induction of melanogenesis was not attributable to inhibition of cyclic nucleotide phosphodiesterase by these compounds. MSH treatment caused a transient rise in cAMP levels (up to 200-fold by 5 min and returning to near basal levels by 5 h). It also stimulated PKA activity up to 5-fold, and the temporal kinetics of this activation mirrored the changes in cAMP levels. In comparison, the PADPRP inhibitors had no effect on either of these processes. These data constitute a novel demonstration of a cAMP-independent mechanism for the induction of melanoma cell differentiation, including melanogenesis.


P2Y purinoceptors in normal NIH 3T3 and in NIH 3T3 overexpressing c-ras. The ability of purinergic agonists to induce Ca2+ responses has been tested in two lines of murine fibroblasts: normal NIH 3T3 fibroblasts and NIH 115.14, a clone expressing high levels [1] of the c-ras protooncogene. Both kinds of cells are responsive to ATP in the range 1 microM-1 mM; ADP and ATP gamma S are almost as potent as ATP, while AMP is unable to elicit a response. Ca2+ measurements performed in single cells by image analysis show great variability among cells but in each individual responding cell the Ca2+ rise occurs in an all-or-none fashion. The transient Ca2+ response does not depend on influx from the extracellular medium. Electrophysiological experiments reveal the activation of an outward current (at -50 mV) by ATP, probably due to Ca(2+)-activated K+ channels, confirming the absence of a substantial Ca2+ influx. Finally, stimulation by ATP produces a small but significant increase in the production of inositol phosphates. These results indicate that these cell lines possess purinergic receptors which are not integral membrane channels and which are coupled to InsP3 formation and may be therefore classified as P2Y.


Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species. Hepatocyte growth factor (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell growth and motility and analyzed the HGF receptor in various species of cells. HGF stimulated growth and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their growth. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with Kd values of 23 and 260 pM, respectively. High-affinity HGF receptor with Kd values of 20-25 pM was detected at 40-720 sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in fibroblasts and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Northern blots revealed that cells possessing a high-affinity HGF receptor expressed c-MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell growth and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.


Fibronectin gene expression in proliferating, quiescent, and SV40-infected mouse kidney cells. To study alterations in cellular gene expression in mouse kidney cell cultures infected with simian virus 40 (SV40) or polyomavirus, we performed a differential screening of a mouse kidney cDNA library with probes prepared from mRNAs of virus-infected and mock-infected cells. We isolated and characterized cDNA recombinant pKT13 which detected increased mRNA levels in infected cells. Sequence analysis of pKT13 revealed close to 100% homology with the 3'-end of mouse fibronectin (FN) mRNA. Since primary cultures of baby mouse kidney cells have been extensively characterized in our laboratories, we studied FN gene expression at different stages of uninfected and virus-infected cultures. High levels of FN and of its mRNA were found in the kidneys of suckling mice, while in primary cultures of proliferating epithelial kidney cells the expression of FN was very low until the cultures became confluent. Thereafter FN increased and reached high levels in cells which were irreversibly arrested in phase Go and which had apparently exhausted their finite division potential. Infection of confluent cultures with polyomavirus or SV40 resulted in a further stimulation of FN gene expression. However, during abortive infection with SV40, FN mRNA and FN levels decreased with emergence of transformed cells and were low in an established SV40-transformed mouse kidney cell line. These changes in FN gene expression suggest that high levels of FN might be indicative in vivo for terminal differentiation and in vitro for cellular senescence.


Restriction enzyme banding and in situ nick-translation on different types of hetero- and euchromatin. We studied the role of chromatin accessibility and methylation in the banding patterns produced by means of in situ nick-translation (NT) and restriction enzyme (RE) banding techniques. For these studies we used the X chromosomes of Microtus cabrerae because of their large segment with four different types of constitutive heterochromatin and because in these chromosomes we can also compare active and inactive euchromatin. The results demonstrate that constitutive heterochromatin in the X chromosomes of M. cabrerae is methylated at specific sequences in both active and inactive Xs. They also show that NT-based techniques are suitable for detecting weak differences in chromatin accessibility, such as differences between active and inactive euchromatin, and are able to distinguish methylation only at the accessible sites. Thus, when methylation has to be mapped in situ, additional experiments have to be performed in order to distinguish findings due to differential accessibility. RE banding seems less sensitive to slight differences in chromatin accessibility, and might thus be more suitable than in situ NT-based techniques for methylation mapping. In harmony with these results, HpaII-based RE banding is able to distinguish between active and inactive euchromatin, possibly depending on its methylation status.


Identification of human and bovine rotavirus serotypes by polymerase chain reaction. The use of the polymerase chain reaction (PCR) for identifying serotypes of human and bovine rotaviruses was examined. In the identification of 115 human rotavirus samples in stools, results with PCR showed excellent agreement with results of serotyping by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies. Furthermore, the PCR showed a much higher sensitivity (93%) than the ELISA test (82.6%). The PCR method could also be applied for identifying the serotype of bovine rotaviruses.


D600 increases the resistance associated with the equatorial potassium current of the lens. The effects of D600, the methoxy analog of verapamil, on a pCMPS system were studied. A major effect of D600 is to increase the resistance associated with the equatorial potassium current of the lens. The increase in resistance is statistically significant at concentrations above 200 microM. At concentrations of 25-50 microM, D600 counteracts the decrease in resistance produced by binding sulfhydryl groups with 1 microM pCMPS. This effect is similar to that produced by quinine and by a calcium-free medium, and is attributed to the prevention of an increase in the calcium-dependent conductance produced by pCMPS.


Coactivation of metabotropic glutamate receptors and of voltage-gated calcium channels induces long-term depression in cerebellar Purkinje cells in vitro. Using an in vitro slice preparation, we studied the effects, on parallel fiber (PF)-mediated EPSPs, of coactivation of metabotropic-glutamate receptors and of voltage-gated calcium (Ca) channels of Purkinje cells (PCs) by bath application of 50 microM trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD) and by direct depolarization of the cells, respectively. These effects were compared with changes in synaptic efficacy obtained when alpha-amino-3hydroxy-5-methylisoxalone-4-propionate (AMPA) receptors of PCs were also activated through stimulation of PFs during the pairing protocol, as well as when similar experiments were performed without trans-ACPD in the bath. In a control medium, pairing for 1 min of PF-mediated EPSPs evoked at 1 Hz with Ca spikes evoked by steady depolarization of PCs (n = 13) led to LTD of synaptic transmission in 9 cases whereas for the others EPSPs were not affected. No LTD occurred in 9 out of 10 other cells tested when PF stimulation was omitted during the 1 min period of Ca spike firing of PCs. Bath application of 50 microM trans-ACPD, in conjunction with the same pairing protocol as before (n = 8), led to a significantly larger LTD of PF-mediated EPSPs after washing out of this drug. Moreover, a clear-cut LTD of PF-mediated EPSPs was also observed in 5 of the 8 other cells, when PF stimulation was omitted during Ca spike firing in the presence of trans-ACPD.(ABSTRACT TRUNCATED AT 250 WORDS)


Lipid peroxidation and myocardial vulnerability in hypertrophied SHR myocardium. In a comparison using age-matched Wistar-Kyoto rats (WKY), 16-week-old male spontaneously hypertensive rat (SHR) hearts were examined histologically and biochemically on the first and fourth day after administration of 20 mg/kg doxorubicin in order to examine whether membrane abnormalities in hypertrophied SHR myocardium are caused by lipid peroxidation. Morphological examination of the SHR revealed focal myocytolysis on the first day and severe cardiomyopathy involving diffuse myocytolysis and vacuolar degeneration in the left ventricle on the fourth day. The activity of a membrane-related enzyme, Na+/K(+)-ATPase, was already lower in control SHR than that of control WKY and was lower in both SHR and WKY than in the respective saline groups on the first day after administration, whereas the enzyme activity in the doxorubicin-treated SHR was not significantly different from that of the treated WKY. A thiobarbituric acid-reactant substance, a lipid peroxidation marker, was significantly higher in treated SHR than it was in the treated WKY on the first day. Furthermore, in comparison with WKY, alpha-tocopherol in the left ventricle in SHR was significantly lower on the fourth day after administration. These results show that a proneness to lipid peroxidation in the membrane system is closely associated with severity of doxorubicin-induced cardiomyopathy in SHR and suggests that membrane lipid peroxidation may cause a higher degree of vulnerability in hypertrophied SHR myocardium.


In vivo formation of polyphosphoinositide isomers and association with progression of murine polycystic kidney disease. Polyphosphoinositide isomers have been demonstrated to be important mediators of cell proliferation in vitro. The present study demonstrates, for the first time, the in vivo formation of the novel isomer, phosphatidylinositol(3)phosphate, in the kidney and liver of intact animals following intraperitoneal administration of [3H]myo-inositol. The formation of renal [3H]phosphatidylinositol(3)phosphate relative to total [3H]phosphatidylinositol-phosphate was positively correlated with cyst proliferation and renal enlargement in a murine model of polycystic kidney disease. Furthermore, despite no difference in the formation of renal [3H]phosphatidylinositol(4)phosphate, a markedly lower accumulation (by 48%) of [3H]phosphatidylinositol(4,5)bisphosphate was observed in the diseased animals as compared to controls. These results indicate that further studies on the in vivo formation of specific polyphosphoinositide isomers in disease states characterized by abnormal growth and oncogene expression are warranted.


Comparative study of six antifungal treatments in an experimental model of murine cryptococcosis. A comparative study, using 5 antifungal drugs for the treatment of an experimental model of murine cryptococcosis, was carried out. One hundred and eighty Balb C mice, divided in 18 groups of 10 animals each, were intraperitoneally inoculated with 10(7) cells of Cryptococcus neoformans var. neoformans. Twelve groups were treated with different schedules beginning 5 days after inoculation, for 2 or 4 weeks. The treatments were the following: amphotericin B (6 mg/kg/every other day, intraperitoneally); 5-fluorocytosine (300 mg/kg/day, by gavage); amphotericin B (6 mg/kg/every other day, intraperitoneally) in association with 5-fluorocytosine (300 mg/kg/day, by gavage); fluconazole, itraconazole and Sch 39.304 (all at the daily dose of 16 mg/kg, by gavage). The six remaining groups were used as controls and received the solvent for the drugs. The evaluation of the efficacy of the different treatments was based on: survival time; macroscopy of brain, lungs, liver and spleen at autopsies; presence of encapsulated yeasts in microscopic examination of wet preparations of these organs; and cultures of a concentrated suspension of brain and lungs. In the animals treated for 2 weeks, the combination of amphotericin B + 5-fluorocytosine was the most useful; it negativized the micro and macroscopic findings as well as 90% of the cultures, and prolonged the survival time up to 60 days. Sixty per cent of the mice which received amphotericin B exhibited the same survival time and macroscopic findings as those treated with the association of amphotericin B + 5-fluorocytosine. Among the azolic compounds, Sch 39.304 proved to be the most effective in the prolongation of survival time.(ABSTRACT TRUNCATED AT 250 WORDS)


Development of antibody to hepatitis C virus (HCV) in acute and chronic non-A, non-B post-transfusion hepatitis. The antibody to hepatitis C virus (anti-HCV) was measured by an immunoassay in 507 serum samples from 94 patients with acute and chronic post-transfusion non-A, non-B hepatitis (NANB) and in 436 healthy blood donors. Anti-HCV was found in 70.8 of patients with acute hepatitis, in 78.2 with chronic hepatitis, and in 1.4 of healthy blood donors. In acute hepatitis, anti-HCV appeared in the serum from 4 to 34 weeks after transfusion and from 1 to 30 weeks after the onset of the overt disease. Three patients with resolving hepatitis (21%) and 2 who developed chronic hepatitis (10%) lost anti-HCV during a mean follow-up period of 28 months. Among the 36 patients with chronic hepatitis, 2 (6%) lost anti-HCV after 12 months and 8 years respectively. These data indicate that in recent years HCV has been the major etiologic agent of acute and chronic transfusion-associated hepatitis (TAH) in our geographical area. The late appearance of anti-HCV from the onset of clinical and biochemical signs of acute hepatitis in more than 70% of patients limits the diagnostic utility of this assay for an earlier serological diagnosis of acute NANB hepatitis. Additional studies are required to determine the diagnostic significance of this antibody in chronic NANB hepatitis.


Antiviral nucleoside toxicity in canine bone marrow progenitor cells and its relationship to drug permeation. The most promising nucleoside analogs that are currently undergoing preclinical and clinical testing for anti-HIV activity belong to the dideoxynucleoside group. We have studied the toxicity of 3'-azido,3'-deoxythymidine (AZT), 2',3'-dideoxycytidine (DDC), and 2',3'-dideoxyinosine (DDI) in canine bone marrow progenitor cells in culture. AZT potently inhibited both canine CFU-GM and CFU-E with IC50 values of 2 and 8 mumol/l respectively, while DDC was relatively non-toxic to either progenitor with IC50 of > 200 mumol/l and 80 mumol/l respectively. DDI was mildly toxic to the bone marrow progenitors, with IC50 values of 62 mumol/l for CFU-GM and 70 mumol/l for CFU-E. Dipyridamole, a nucleoside transport inhibitor, did not influence the toxicity of these dideoxynucleosides in either progenitor at concentrations up to 10 mumol/l. Using uridine as the prototype endogenous nucleoside, we have demonstrated that there is a saturable "zero-trans" nucleoside transport system in canine bone marrow mononuclear cells, which is completely inhibited by 1 mumol/l dipyridamole (Ki = 0.02 mumol/l). None of the dideoxynucleosides appeared to be a substrate for this transport system, and dipyridamole did not alter their influx. Permeation of radiolabeled AZT into bone marrow mononuclear cells was slow and non-saturable, while the permeation of DDI was even slower. DDC did not permeate bone marrow cells well, with very little cell accumulation even after 2 hours of equilibration. Our toxicity data from canine bone marrow progenitor cells paralleled the clinical hematotoxicity profiles of these dideoxynucleosides in AIDS patients and suggest that the myelotoxicity of a nucleoside analog is related to its ability to permeate the progenitor cells in question. Canine bone marrow progenitor cultures may serve well as an in vitro model for drug hematotoxicity studies.


Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats. Retinoic acid (RA) and triiodothyronine (T3) exert many of their actions by binding to specific nuclear receptors (respectively, RA receptor (RAR) and T3) receptor (TR) belonging to a 'superfamily' of receptors. Some heterologous regulation of these receptors has been shown, and in particular regulation of the maximum binding capacity of TR by either retinol or RA. Now, using hyperthyroidic rats as a model, the effect of RA on binding capacity and on the mRNA levels of TR and RAR was investigated. The results show that the benefit of vitamin A treatment for the hyperthyroidic state, which has been described for a long time, could be the result of a down-heteroregulation of TR by RA, the active metabolite of retinol.


Time-dependent increases in Na+,K(+)-ATPase content of low-frequency-stimulated rabbit muscle. Chronic low-frequency stimulation of rabbit fast-twitch muscle induced time-dependent increases in the concentration of the sarcolemmal Na+,K(+)-ATPase and in mitochondrial citrate synthase activity. The almost twofold increase in Na+,K(+)-ATPase preceded the rise in citrate synthase and was complete after 10 days of stimulation. We suggest that the increase in Na+,K(+)-ATPase enhances resistance to fatigue of low-frequency-stimulated muscle prior to elevations in aerobic-oxidative capacity.


Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.


Isolation and characterisation of a functional alpha beta heterodimer from the ATP synthase of Rhodospirillum rubrum. An alpha beta heterodimer of the F1-ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each alpha beta heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg(2+)-ions. The alpha beta heterodimer restores both ATP-synthetic and -hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol alpha beta.mol BChl-1. The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The Vmax and Km (ATP) for this Mg(2+)-dependent activity were 110 +/- 10 nmol.min-1.mg protein-1 and 100 +/- 30 microM, respectively. The Km did not differ significantly from that of RF1.


Does biocatalysis involve inhomogeneous kinetics? Biocatalytic reactions can occur according to two very different mechanisms: homogeneous, which is described by standard transition state theory (TST) and its modifications, and inhomogeneous (polychromatic), which is characteristic for some of the charge-transfer reactions in liquids and amorphous solids. While most data published on enzyme reactions are interpreted on the basis of homogeneous kinetics, the important recent findings suggest the involvement of inhomogeneous kinetics mechanism.


Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli. A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5 alpha F'). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.


Identification of a small Na+/H+ exchanger-like message in the rabbit myocardium. We examined the Na+/H+ exchanger message in isolated perfused rabbit hearts using Northern blot analysis with cDNA encoding for the rabbit cardiac Na+/H+ exchanger. A cDNA probe from the coding region of the rabbit myocardial Na+/H+ exchanger hybridized to mRNA of 5 kb under high stringency, and to a second 3.8 kb mRNA species under low stringency. When Northern blots were re-probed with a section of the 3'-untranslated region of the cDNA, the 5 kb message was apparent while the smaller 3.8 kb message was not. If isolated working rabbit hearts were subjected to ischemia we observed increases in the 3.8 kb message. Overall, the results show that a 3.8 kb mRNA product, which is homologous to the amiloride sensitive Na+/H+ exchanger, exists in the myocardium and increases during ischemia in the myocardium.


Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae. Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.


Amino acids 356-372 constitute a Gi-activator sequence of the alpha 2-adrenergic receptor and have a Phe substitute in the G protein-activator sequence motif. The human alpha 2-adrenergic receptor contains the sequence KASRWRGRQNREKRFTF (amino acids 356-372) at the C-terminal end of its third intracellular loop. This sequence satisfies the structural criteria for G protein-activating sequences [(1992) J. Biol. Chem. 267, 8342-8346] except that the C-terminal sequence is B-B-X-X-Phe instead of B-B-X-B or B-B-X-X-B (B: basic residue, X: non-basic residue). Nevertheless, the synthetic peptide corresponding to this sequence (peptide alpha 2-F) was found to activate Gi and Go strongly with a saturated effect at 1-3 microM. Furthermore, the substitution of the C-terminal Phe of peptide alpha 2-F with Arg, Trp, and Tyr (but not Ala or Asp) did not appreciably affect the Gi-activating potency. It is suggested that the C-terminal basic residue of the B-B-X-X-B motif in Gi-activating sequences can be replaced by an aromatic residue.


Regulation of 1 alpha, 25-dihydroxyvitamin D3 synthesis in macrophages from arthritic joints by phorbol ester, dibutyryl-cAMP and calcium ionophore (A23187). Phorbol 12-myristate 13-acetate (100 nM), a potent protein kinase C and macrophage activator, has a biphasic affect on 25(OH)D3-1 alpha-hydroxylase activity in synovial fluid macrophages from arthritis patients. After 5 h, 1 alpha, 25(OH)D3 synthesis fell from 5.2 +/- 0.1 to 1.6 +/- 0.2 pmol/h per 10(6) cells, however, after 24 h and 48 h, synthesis increased to 17.4 +/- 0.3 and 22.3 +/- 1.4 pmol/h per 10(6) cells, respectively. Although an independent short-term mechanism is suggested, protein kinase C may promote macrophage activation, thus increasing long-term 25(OH)D3-1 alpha-hydroxylase expression. Intracellular calcium and cAMP are unlikely to activate the enzyme, since 0.1 microM of the calcium ionophore, A23187, and 1 mM dibutyryl-cAMP inhibited synthesis by 87% and 79%, respectively, after 24 h.


Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria. The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.


Regulatory regions in the yeast FBP1 and PCK1 genes. By deletion analysis of the fusion genes FBP1-lacZ and PCK1-lacZ we have identified a number of strong regulatory regions in the genes FBP1 and PCK1 which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. Lack of expression of beta-galactosidase in fusions lacking sequences from the coding regions suggests the existence of downstream activating elements. Both promoters have several UAS and URS regions as well as sites implicated in catabolite repression. We have found in both genes consensus sequences for the binding of the same regulatory proteins, such as yAP1, MIG1 or the complex HAP2/HAP3/HAP4. Neither deletion nor overexpression of the MIG1 gene affected the regulated expression of the FBP1 or PCK1 genes.


Human guanylin: cDNA isolation, structure, and activity. Guanylin is a mammalian peptide homologue of heat-stable enterotoxins that acts on intestinal guanylate cyclase to elicit an increase in cyclic GMP. We have isolated a cDNA encoding an apparent precursor of guanylin from a human intestinal cDNA library. The mRNA is expressed at high levels in human ileum and colon. Human guanylin stimulated increases in T84 cell cyclic GMP levels, displaced 125I-labelled heat-stable enterotoxin (STa) binding to this cell line, and stimulated increases in short-circuit current (Isc) of isolated rat proximal colonic mucosa. This peptide may play a role in regulating fluid and electrolyte absorption in human intestines.


Intracellular signal transduction pathways that control pancreatic beta-cell proliferation. This review focuses on the factors that regulate the proliferation of pancreatic islet beta-cells in vitro, and in particular on the intracellular pathways that convey the mitogenic signal into a proliferative response. Substances as diverse as nutrients, polypeptides, cytokines, adrenergic agents, lithium, phorbol esters and cyclic AMP analogs are all able to stimulate or inhibit beta-cell proliferation in a time- and concentration-dependent manner. The evidence for involvement of cyclic AMP, cyclic GMP, protein kinase C, inositol polyphosphates, GTP-binding proteins, polyamines and oncogenes is reviewed.


Ionization of the heme propionate substituents in pseudomonad cytochromes c-551. The heme propionate substituents in Pseudomonas cytochrome c-551 are partially buried by folds of polypeptide in the structure of the protein, and are involved in several hydrogen bonds. The ionization behavior of these groups has been of interest because the oxidation potential of the heme changes with pH in a manner that may parallel ionization of a propionate. The ionization pKa's of these groups have been determined by following the NMR chemical shifts of nearby protons acting as probes of the ionization state of the propionates. In Pseudomonas aeruginosa c-551 the 13-propionate (IUB-IUPAC porphyrin nomenclature) has been assigned a pKa of 3.1, and the 17-propionate a pKa of 7.2. In the homologous Pseudomonas stutzeri c-551, the respective propionates both have pKa values of 3.0.


Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity. Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.


[Effect of Ca(2+)-channel antagonists on the corticoidogenesis in primary cultured bovine adrenocortical cells]. We investigated the effect of Ca(2+)-channel antagonists, Nicardipine(N), Verapamil(V) and Flunarizine(F), on the corticoidogenesis(CG) in primary cultured bovine adrenocortical cells. To examine the effect on receptor operated Ca(2+)-channel(ROC) and voltage operated Ca(2+)-channel(VOC), involved in corticoid synthesis, adrenocorticotropic hormone(ACTH) and high-K+ were used respectively. With or without the antagonists, cells were incubated at 37 degrees C for 1h in the presence of ACTH (100pM) or K+ (30mM). Corticoid was measured fluorometrically using cortisol as the standard. N and V inhibited not only ACTH-induced CG, but high K(+)-induced CG in a concentration-dependent manner. However, F inhibited only high K(+)-induced CG, and did not affect ACTH-induced CG. These inhibitions were observed at the low micromolar concentrations (below 40 microM) of the antagonists. In the regulation of corticoid synthesis, we indicate that F has an inhibitory effect on VOC without ROC; on the other hand, N and V inhibit both ROC and VOC.


[A case of Cushing syndrome caused by ACTH producing atypical lung carcinoid tumor]. A forty-one-year-old male who was found to have lung carcinoid tumor showed clinical features of Cushing syndrome. At the age of 38 years during a regular check-up, a chest roentgenogram showed multiple nodular shadows in the bilateral lung, and he was admitted to hospital. By open lung biopsy, he was diagnosed as having malignant carcinoid or small cell carcinoma of the lung. The abnormal lung shadows did not respond to chemotherapy (CDDP + VDS). The clinical course after discharge was uneventful until the age of 41 years, when he was readmitted to our hospital because of brain metastasis. Physical examination revealed moon face and central obesity. Plasma ACTH level was high and dexamethasone suppression test showed no cortisol suppression on 8 mg dexamethasone administration. Therefore, it was thought that the patient had ACTH producing ectopic tumor which led to Cushing syndrome. We measured the molecular weight of ACTH by column chromatography and found he had a big ACTH (molecular weight about 22,000). Reexamining him clinically and histologically, we concluded that the patient had atypical carcinoid tumor in the lung which might produce ACTH causing Cushing syndrome with metastasis to the brain.


Materials and techniques in fixed prosthodontics. If we have learned anything from our experiences with all-ceramic systems such as Cerestore, Cerapearl, and even Dicor, it is that it takes years to uncover the strengths, weaknesses, and limitations of new materials and technologies. Manufacturers' initial claims for fit, strength, esthetics, biocompatibility, wear characteristics, and clinical performance should be viewed as preliminary until supported by independent laboratory and clinical studies. Laboratory testing alone may not provide sufficient insight into the relative strengths and weaknesses of products or assure comparable performance in the commercial dental laboratory or the ultimate testing arena, the oral environment. Moreover, the diversity of the new materials and techniques discussed demonstrates the breadth and depth of the technologic changes in dental biomaterials. The rate at which these dental products enter the marketplace and their sheer number alone are indeed staggering. Furthermore, these advances are not emerging from any single nation but from manufacturers all around the world (Japan, Germany, as well as the United States), indicating our international reliance on one another. Clearly, the decade of the 1990s is emerging as an exciting period in the development of biomaterials in comprehensive fixed prosthodontics and dentistry in general.


Clinical and radiological findings in Gardner's syndrome: a case report and follow-up study. Gardner's syndrome is characterized by colorectal adenomas, multiple osteomas, especially of the skull, and various soft-tissue tumours. The disease is inherited as an autosomal dominant disorder and all untreated patients will develop colorectal adenocarcinomas. Since the clinical and radiological stigmas in the maxillofacial area, such as exosteal and endosteal osteomas, skin cysts, atypical skin pigmentation and abnormal dental findings or radiopaque lesions can precede the often symptomless adenomas for many years, dentists, maxillofacial surgeons and radiologists should be familiar with the manifestations of this disease. A case is reported to illustrate the dentist's role in the diagnosis of Gardner's syndrome. In a follow-up study of 11 patients with colorectal adenomas, the typical triad of features of Gardner's syndrome was found in eight. In six patients, bony changes were demonstrated by panoramic radiography. It is proposed that radiography of the jaws may serve as a valuable tool for the early detection of carriers of Gardner's syndrome.


Postnatal expression of the inositol 1,4,5-trisphosphate receptor in canine cerebellum. 1. Inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, has been shown to be the link between activation of several plasma membrane receptors and Ca2+ release from intracellular, membrane-bound compartments. In this study, the postnatal expression of the canine cerebellum IP3 receptor was investigated by biochemical, ligand binding and immunocytochemical methods. 2. Specific receptor sites for IP3 and the extent of IP3-induced Ca2+ release were quantitated in microsomal fractions isolated from cerebella of developing (0-28 day-old) and adult dogs. The IP3 receptor was detected in newborn animals and adult levels were attained within 3-4 weeks. 3. The time-course of IP3 receptor ontogeny paralleled both growth of Purkinje neurons, as indicated by immunofluorescence of cerebellum cortex cryosections with anti-IP3 receptor antibodies, and synaptogenesis, as judged by Western blotting of the microsomal fractions with anti-synaptophysin antibodies.


Increase in superoxide production by heat-shocked cells of Neurospora crassa, demonstrated by a fluorometric assay. 1. Increase in superoxide production by heat-shocked cells of Neurospora crassa was demonstrated by a fluorometric assay. 2. A sensitive fluorometric assay for the estimation of superoxide anion radical--based on the liberation of 4-methylumbelliferone from 4-methyl-beta-D-umbelliferyl glucopyranoside--is described. 3. Using this system the level of superoxide in the medium of heat-shocked Neurospora crassa cells was found to be consistently higher, in comparison with that of non-shocked cells, cultured at the normal growth temperature of 28 degrees C. 4. Addition of superoxide dismutase to the culture media suppressed the production of 4-methylumbelliferone.


On the specificity of dolichol kinase and DolPMan synthase towards isoprenoid alcohols of different chain length in rat liver microsomal membrane. 1. A wide range of dolichols differing in the length of hydrocarbon chain (from 11 to 32 isoprene residues) were found to be phosphorylated in the presence of CTP in rat liver microsomes. 2. Fully unsaturated polyprenols of the same chain length as dolichols were poor substrates for dolichol kinase at low detergent (Nonidet P-40) concentration. At higher concentration of detergent, both dolichols and polyprenols were equally effective. 3. In the transfer of mannosyl residues from GDPMan, the dolichyl phosphates generated in rat liver microsomes were all good lipid acceptors, while fully unsaturated polyprenyl phosphates were not.


Hypothesis: low Na/K-ATPase activity in the red cell membrane, a potential marker of the predisposition to diabetic neuropathy. The development of diabetic complications does not depend entirely on diabetes duration and control. Predisposing and aggravating factors, either constitutional or environmental, seem to play a role. We have previously observed that polyneuropathy is more frequent, of earlier onset, and more severe among North African insulin-dependent diabetic patients than among Europeans matched for sex, duration and control of diabetes. The Na/K-ATPase activity displays sex and ethnic differences and a dysfunction of this enzyme is probably involved in the pathogenesis of diabetic neuropathy. We have therefore postulated that the predisposition of some diabetic patients to develop a polyneuropathy could be related to a low Na/K-ATPase activity. Red cell membrane Na/K-ATPase activity was studied in European men presenting with insulin-dependent diabetes mellitus for more than 15 years. 10 patients with neuropathy were matched to 10 patients void of neuropathy on duration of diabetes and HbA1c values. Thirteen healthy European men and 13 North African men born and living in France were also studied. Na/K-ATPase activity was lower in patients with neuropathy (200 +/- 31 vs 289 +/- 42 nmol Pi.mg protein-1.h-1 mean +/- SD; p less than 0.05). When compared to that of 13 European healthy men, Na/K-ATPase activity was lower in the whole group of diabetic patients but appeared to be in the normal range for patients without neuropathy and decreased in those with neuropathy. The 13 North African healthy men had lower values than the European healthy men (227 +/- 46 vs 298 +/- 60 nmol Pi.mg protein -1.h-1, p less than 0.05). Red cell membrane Na/K-ATPase activity is low in insulin dependent patients with neuropathy compared to those without neuropathy. This finding probably reflects a constitutional difference. This association could be explained if red cell and nerve membrane Na/K-ATPase abnormalities behave similarly according to glycaemic control in diabetic subjects. It is suggested that the predisposition to neuropathy of North African insulin-dependent diabetic patients may be related to lower Na/K-ATPase activity. A high level of activity of this enzyme may protect from diabetic neuropathy. This enzyme activity could be a marker of predisposition towards diabetic polyneuropathy.


Variations in Ca(2+)-mediated activation of Ca(2+)-ATPase and its associated inhibitor in erythrocyte membrane. 1. Two distinct patterns of Ca(2+)-mediated activation of Ca(2+)-ATPase were identified in calmodulin-depleted membranes. 2. In membranes showing no activation (type A), preincubation with micromolar concentration of cyclic AMP and ATP made possible stimulation of the enzyme while in membranes already exhibiting activation (type B), preincubation with cyclic AMP and ATP abolished the activation. 3. ATPase stimulation in type A membranes was suppressible by leupeptin. 4. Triton extractable inhibitor isolated from type A membranes was as active as that derived from type B membranes only after preincubating the membranes with cyclic AMP and ATP. 5. The inhibitor could be inactivated by alkaline phosphatase.


Cyclic AMP and the regulation of cholesterol metabolism. Cyclic AMP has been implicated to a greater or lesser extent in the regulation of four key enzymes which interact to regulate intracellular cholesterol metabolism; HMG CoA reductase; ACAT; cholesteryl ester hydrolase; and cholesterol 7 alpha hydroxylase. The relationship between these enzymes and the sites where current evidence suggests that cyclic AMP may be involved are summarized in Fig. 3. Cholesterol 7 alpha hydroxylase controls the catabolism of cholesterol to bile acids in the liver, and thus its removal from the body via the bile, but does not have a major role in cholesterol metabolism in extrahepatic tissues. It is clear that cyclic AMP is able to influence the activity of this enzyme in liver sub-cellular fractions and isolated hepatocytes in vitro, and studies in our laboratory have shown that changes in Ca2+ fluxes within the cell may be important in its mechanism of action. Whether or not the cyclic nucleotide has a role regulating cholesterol 7 alpha hydroxylase activity in vivo, however, is not known. HMG CoA reductase is inactivated by phosphorylation both in vitro and in vivo, but although cyclic AMP and glucagon have been shown to inhibit the enzyme, cyclic AMP-dependent protein kinase is not directly involved. The exact mechanism by which the cyclic nucleotide influences the system remains unclear, but it may be related to activation of microsomal phosphatases. The activity of ACAT has been shown to be modulated by phosphorylation in a number of tissues in vitro, but the involvement of cyclic AMP has not been unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)


Inositol lipid hydrolysis contributes to the Ca2+ wave in the activating egg of Xenopus laevis. We have used fluorescence ratio-imaging of fura-2 in the activating egg of Xenopus laevis to study the wave of increased intracellular free Ca2+ concentration ([Ca2+]i) while monitoring that of cortical granule exocytosis. Naturally matured eggs were dejellied, injected with fura-2, and activated by the iontophoresis of 1-30 nCoul of inositol-1,4,5-trisphosphate which triggers an immediate increase in free [Ca2+]i at the injection site. The Ca2+ rise spreads throughout the egg, reaching the opposite side in 5-8 min, and is followed by elevation of the fertilization envelope about 20-30 sec behind the [Ca2+]i wave. [Ca2+]i returns to preactivation levels within about 20 min after activation. We further studied the role of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by microinjecting antibodies to PIP2 into the egg. PIP2 antibodies did not alter the propagation velocity of the wave but greatly reduced the amount of Ca2+ released in the egg cortex. These data suggest that PIP2 hydrolysis plays a role in the release of [Ca2+]i in the outer regions of the egg following activation.


Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy. IDDM patients with incipient and overt nephropathy have been found to exhibit an overactivity of RBC sodium-lithium countertransport. To explore the physiological relevance of this finding, we measured the activity of Na+/H+ antiport in serially passaged cultured skin fibroblasts from IDDM patients with and without nephropathy and from normal, nondiabetic control subjects. Na+/H+ antiport activity (measured as the rate of amiloride-sensitive Na+ influx at pHi = 6.4, extracellular pH = 8.0, and [Na+] = 1 mM) was elevated significantly in IDDM patients with nephropathy compared with IDDM patients without nephropathy and nondiabetic control subjects (13.35 +/- 3.8 vs. 8.54 +/- 2.0 vs. 7.33 +/- 2.3 nmol Na+.mg protein-1.min-1; P less than 0.006 and P less than 0.001, respectively). A kinetic analysis of Na+/H+ antiport activity showed that the raised activity in IDDM patients with nephropathy was caused by an increased Vmax for extracellular Na+. Km values were similar in the three groups. pH-stimulated amiloride-sensitive Na+ influx also was higher under baseline conditions and after serum stimulation in cells from IDDM patients with nephropathy. pHi values were significantly higher, both during active proliferation and after 10-min exposure to serum, in cells from IDDM patients with nephropathy, compared with IDDM patients without nephropathy and nondiabetic control subjects. Serum-stimulated incorporation of [3H]thymidine into DNA was greater in IDDM patients with nephropathy than in the other two groups (35.7 +/- 18.9- vs. 17.4 +/- 7.5- vs. 11.9 +/- 8.7-fold stimulation above baseline; P less than 0.01 for both.(ABSTRACT TRUNCATED AT 250 WORDS)


Mutated insulin receptor Val996 reduces insulin-dependent generation of inositol glycan and diacylglycerol. We evaluated whether insulin-receptor tyrosine kinase activity is required for activation of PDH, insulin-induced hydrolysis of PIG and generation of IG and 1,2-DAG. For the analysis, we used stable-transfected CHO cell lines expressing wild-type human insulin receptor (CHO-wt cells) or the mutant receptor (Val996) that lacks tyrosine kinase activity (CHO-mut cells) (1,2). Insulin stimulated PDH activity in three CHO cell lines in a dose-dependent manner. Half-maximal concentrations of insulin to activate PDH was 7 x 10(-11) M in the CHO-wt cells, 10(-9) M in the parental cells, and 8 x 10(-9) M in the CHO-mut cells. Insulin stimulated hydrolysis of PIG and generation of IG and DAG in three CHO cell lines in a dose-dependent manner. Half-maximal concentrations of insulin to induce generation of IG was 8 x 10(-11) M in the CHO-wt cells, 10(-9) M in the parental CHO cells, and 10(-8) M in the CHO-mut cells. ED50 for the stimulation of DAG generation was 7 x 10(-11) M in the CHO-wt cells, 10(-9) M in the parental cells, and 10(-8) M in the CHO-mut cells. It is concluded that insulin-dependent PDH activation, PIG hydrolysis, and IG and DAG generation are mediated by the wild-type but not by the mutated insulin receptor of Val996. This study suggests that tyrosine kinase activity of the insulin receptor might be a prerequisite for insulin-stimulated generation of IG and DAG.


Lilly Lecture: molecular mechanisms of insulin resistance. Lessons from patients with mutations in the insulin-receptor gene. Insulin resistance contributes to the pathogenesis of NIDDM. We have investigated the molecular mechanisms of insulin resistance in patients with genetic syndromes caused by mutations in the insulin-receptor gene. In general, patients with two mutant alleles of the insulin-receptor gene are more severely insulin-resistant than are patients who are heterozygous for a single mutant allele. These mutations can be put into five classes, depending upon the mechanisms by which they impair receptor function. Some mutations lead to a decrease in the number of insulin receptors on the cell surface. For example, some mutations decrease the level of insulin receptor mRNA or impair receptor biosynthesis by introducing a premature chain termination codon (class 1). Class 2 mutations impair the transport of receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane. Mutations that accelerate the rate of receptor degradation (class 5) also decrease the number of receptors on the cell surface. Other mutations cause insulin resistance by impairing receptor function--either by decreasing the affinity to bind insulin (class 3) or by impairing receptor tyrosine kinase activity (class 4). The prevalence of mutations in the insulin receptor gene is not known. However, theoretical calculations suggest that approximately 0.1-1% of the general population are heterozygous for a mutation in the insulin-receptor gene; the prevalence is likely to be higher among people with NIDDM. Accordingly, it is likely that mutations in the insulin-receptor gene may be a contributory cause of insulin resistance in a subpopulation with NIDDM.


Suspect value of non-CSF viral cultures in the diagnosis of enteroviral CNS infection in young infants. Laboratory criteria used for presumptive diagnosis of enteroviral meningitis were evaluated as predictors of cerebrospinal fluid (CSF) infection. Records were retrospectively analysed of infants under four months of age admitted to hospital between 1977 and 1987 with viral CSF cultures: those with enteroviruses isolated from CSF (group 1) were compared with those with enteroviruses isolated only from non-CSF sites (group 2). Predictive value computations demonstrated that no single or combined non-CSF culture accurately predicted isolation of enteroviruses from the CSF. These results suggest that CSF viral culture is imperative in establishing the diagnosis of enteroviral meningitis in young infants.


The preclinical safety evaluation of human monoclonal antibody against cytomegalovirus. The human monoclonal antibody against cytomegalovirus (Mab C23) was examined pharmacokinetically and toxicologically as part of the preclinical studies prior to approval for human use. Rats given repeated intravenous administrations of Mab C23 produced no antibodies against Mab C23 and maintained a blood Mab C23 level in a dose-dependent manner. However, pregnant rabbits produced antibodies against Mab C23. The half-life of Mab C23 in plasma was 15.9 days in rats, which was similar to that of normal human serum gamma-globulin (NHSG). Neither behavioral effects nor circulatory disturbance was found in mice, rats, and dogs even after a single intravenous injection of 100 or 200 mg/kg, which corresponds to 50 or 100 times the intended clinical dosage. The repeated doses of 2, 10, or 20 mg/kg of Mab C23 on six occasions with 1- or 2-week intervals elicited a transient decrease in leukocyte counts in rats given 10 or 20 mg/kg, but no adverse effects in cynomolgus monkeys. Mab C23 did not cause any reproductive or developmental toxicity when administered to rats and rabbits at dose levels of 20 mg/kg or less. However, pregnant animals showed lower plasma levels of Mab C23 than non-pregnant animals. The chromosomal aberration test disclosed no clastogenicity in human lymphocytes. An immunostaining for Mab C23 revealed no localizations in several tissues of cynomolgus monkeys given intravenous doses of Mab C23.(ABSTRACT TRUNCATED AT 250 WORDS)


Lipoprotein receptors, plasma cholesterol metabolism, and the regulation of cellular free cholesterol concentration. Classical concepts of the regulation of plasma cholesterol levels involve roles for the "forward" delivery of low density lipoprotein (LDL) cholesterol from the liver to the peripheral tissues, mediated by the LDL receptor, and a "reverse" delivery of cholesterol in the form of high density lipoprotein (HDL) from the peripheral tissues to the liver. Candidate receptors for HDL in peripheral tissues and for chylomicrons in the liver have more recently been described, and a receptor of uncertain function recognizing chemically modified LDL has also been identified. The activities of all the well-characterized lipoprotein receptors, as well of major catalytic factors in plasma that regulate cholesterol esterification and cholesteryl ester transfer between lipoproteins, reflect the need to maintain plasma membrane free cholesterol level, and its direct and indirect effects within the membrane, within well-defined limits.


Acute experimental esophagitis impairs signal transduction in cat lower esophageal sphincter circular muscle. It has been previously shown that induction of experimental esophagitis in the cat by esophageal perfusion for 30 minutes with 0.1N HCl for 4 consecutive days results in a significant reduction of in vivo lower esophageal sphincter (LES) resting pressure and in vitro spontaneous tone without affecting esophageal response to KCl. It has also been shown that basal LES tone and LES contraction in response to acetylcholine depend on the release of calcium from intercellular stores, whereas esophageal contraction is mediated by extracellular calcium. The present report shows that esophageal acid perfusion impairs the transduction pathway mediating lower esophageal sphincter contraction in response to acetylcholine through release of intracellular calcium because LES strips and single cells no longer contract in response to acetylcholine if calcium is removed from the physiologic salt solution. This suggests that either the intracellular calcium stores or the release mechanisms that mediate maintenance of tone and contraction in response to acetylcholine may be damaged. However, the acid perfusion has no effect on the acetylcholine response in the esophagus, which is mediated by the influx of extracellular calcium. In the LES circular muscle, the injury results in reduced levels of inositol phosphates without affecting resting levels of 5'-cyclic adenosine monophosphate or 5'-cyclic guanosine monophosphate. The reduced levels of 1,4,5-inositol trisphosphate are consistent with impairment in the mechanisms responsible for release of intracellular calcium, although concurrent damage to calcium stores may also occur.