The role of adenosine in glycine-induced glomerular hyperfiltration in rats. Ingestion of a high-protein diet or infusion of amino acids induces glomerular hyperfiltration and hyperemia. We have investigated the role of endogenous adenosine in glycine-induced hyperfiltration. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious chronically instrumented rats. Glycine (3.7 mg/min, i.v.; n = 6) significantly increased GFR and ERPF from 0.92 +/- 0.07 to 1.13 +/- 0.08 and 3.28 +/- 0.24 to 3.69 +/- 0.19 ml/min.100 g, respectively. In the presence of adenosine deaminase (ADA, 2 U/kg.min, n = 6), glycine-induced glomerular hyperfiltration and hyperemia were blunted. The small changes in GFR (from 0.86 +/- 0.06 to 0.90 +/- 0.10 ml/min.100 g) and ERPF (from 3.60 +/- 0.57 to 3.83 +/- 0.53 ml/min x 100 g) were not statistically significant. Erythro-9-(2-hydroxy-3-nonyl) adenosine hydrochloride (100 micrograms/kg.min, n = 6), an ADA inhibitor, reversed the effect of ADA. Injection of 8-phenyltheophylline (10 mg/kg, n = 6), an adenosine A1 receptor antagonist that alone did not affect GFR, abolished the glycine-induced glomerular hyperfiltration (GFR from 1.02 +/- 0.08 to 0.93 +/- 0.08 ml/min.100 g, P > .05). 8-phenyltheophylline, which itself decreased ERPF, also significantly decreased the ERPF response to glycine (3.47 +/- 0.26 to 2.78 +/- 0.14 ml/min x 100 g). Thus, endogenous adenosine, acting at adenosine A1 receptors, plays an important role in the glomerular hyperfiltration and hyperemia induced by glycine.


Evidence for the interaction between antacid and gastric mucosa using an "artificial stomach" model including gastric mucosa. In light of evidence that certain aluminum-based antacids adhere to the gastric mucosa, we modified our previously described "artificial stomach" (AS) model by including a piece of hog stomach and compared the antacid activity of six aluminum-containing antacid products in the model with and without gastric mucosa. The activity of three of these, Maalox, Riopan and Supralox, was not significantly different in the two systems. In contrast, the activity of the other three, Aludrox, Phosphalugel and Simeco, was significantly greater with mucosa. Antacid activity of one product from each set (Supralox, Phosphalugel) was evaluated in two in vivo methods in human volunteers. For both antacids, results in vivo were similar to those obtained with the AS-containing mucosa. Without mucosa, in vivo and in vitro results were dissimilar for Phosphalugel, thus validating the modified AS. The difference between the two sets of antacids can be explained by 1) the fact that the Al:Mg ratio in the set affected by mucosa is greater than that of unaffected antacids, and 2) a weaker antacid load than in unaffected Supralox. We suggest that in an acid milieu, aluminum ions in antacids like Aludrox, Phosphalugel and Simeco are bound to sialic acid residues in mucus glycoproteins, thus retarding the transit of these antacids through both the AS and the real stomach and prolonging their activity in both situations. When the Al:Mg ratio is low or when the amount of antacid salts is large, aluminum ions tend to be buried in complexes, giving them less chance to interact with gastric mucus, so they transit the stomach more quickly.


Neuropeptide Y potentiates selectively the N-methyl-D-aspartate response in the rat CA3 dorsal hippocampus. I. Involvement of an atypical neuropeptide Y receptor. Neuropeptide Y (NPY) has been reported to potentiate N-methyl-D-aspartate (NMDA)-induced neuronal activation in the rat CA3 region of the dorsal hippocampus in vivo. Three types of NPY receptors, denoted Y1, Y2 and Y3, have been identified thus far. The present studies were undertaken to characterize the type of NPY receptor involved in this effect of NPY on the neuronal response to NMDA. NPY, its analogs [Leu31, Pro34]NPY and desamido-NPY, the related peptides pancreatic polypeptide (PP) and peptide YY (PYY) and the C- and N-terminal NPY fragments, NPY2-36, NPY11-36, NPY13-36, NPY16-36, NPY18-36 and NPY1-24CONH2, were tested. The peptides NPY (which is active at Y1, Y2 and Y3 receptors), [Leu31, Pro34]NPY (a selective Y1 agonist) and NPY13-36 (which mimics the effects of NPY in Y2 models) dose dependently enhanced NMDA-induced activation of CA3 dorsal hippocampus pyramidal neurons, but did not alter the activation of the same neurons by quisqualate. In contrast, PYY (which mimics NPY on Y1 and Y2 receptors, but has no activity or elicits an effect opposite to that of NPY in Y3 models) and NPY18-36 (which has been reported to exert an antagonistic or a partial agonistic action at Y3 receptors) did not modify by themselves the NMDA response, but antagonized the potentiating effect of NPY on NMDA-induced activation. Additionally, the C-terminal desamido form of NPY, which has little or no activity at Y1 and Y2 receptor subtypes, reduced the neuronal response to NMDA and quisqualate.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuropeptide Y potentiates the N-methyl-D-aspartate response in the CA3 dorsal hippocampus. II. Involvement of a subtype of sigma receptor. In an in vivo electrophysiological paradigm, we have shown in the companion paper that neuropeptide Y (NPY) potentiates N-methyl-D-aspartate (NMDA)-induced neuronal activation via a non-Y1, non-Y2, non-Y3 receptor subtype, in the rat CA3 dorsal hippocampus. Because sigma ligands have also been shown to potentiate NMDA-induced activation and because NPY and peptide YY have been reported to have high affinity for sigma binding sites, the present study was carried out to assess the possibility that the modulation of the NMDA response by NPY might be mediated by a sigma receptor. In the same electrophysiological paradigm, low doses of haloperidol and alpha-(4-fluorophenyl)-4-(5-fluoro-2- pyrimidinyl)-1-piperazine butanol, two antagonists of sigma receptors, reversed the potentiation of the NMDA response induced by NPY, [Leu31, Pro34]NPY or NPY13-36 and blocked the suppressant effect of desamido-NPY on the NMDA response. In contrast, spiperone, which has low affinity for sigma sites, was ineffective in suppressing NPY, as well as desamido-NPY-induced modulation of the NMDA response. In our model, peptide YY, which acts as a NPY antagonist by suppressing the potentiation of the NMDA response induced by NPY, also antagonized the potentiation of the NMDA response induced by the administration of low doses of di(2-tolyl)guanidine and (+)N-cyclopropyl-methyl-N-,methyl-1,4- diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride, two high-affinity sigma agonists.(ABSTRACT TRUNCATED AT 250 WORDS)


Involvement of adrenergic and angiotensinergic receptors in the paraventricular nucleus in the angiotensin II-induced vasopressin release. The role of hypothalamic paraventricular adrenoceptors and angiotensin II (ANG II)-AT 1 receptors in mediating the vasopressin (AVP) release into the plasma in response to i.c.v. and local paraventricular ANG II injections was investigated in conscious chronically instrumented rats. Noradrenaline (NA) administered bilaterally into the paraventricular nucleus (PVN) dose-dependently stimulated AVP release. Bilateral PVN microinjections of the alpha 1 adrenoceptor agonists methoxamine and phenylephrine, or of the alpha2 adrenoceptor agonist clonidine, did not affect plasma AVP when given alone, but increased plasma AVP when methoxamine and clonidine were given in combination. In contrast, PVN microinjections of both the beta 1 adrenoceptor agonist dobutamine and the beta 2 adrenoceptor agonist salbutamol significantly reduced basal plasma AVP. Bilateral PVN pretreatment with the alpha 1 and alpha 2 adrenergic antagonists prazosin, idazoxan and rauwolscine, but not of the beta 1 and beta 2 adrenoceptor antagonists atenolol and ICI 118 551, significantly attenuated the i.c.v. ANG II-induced AVP release. ANG II injected bilaterally into the PVN dose-dependently increased plasma AVP. Bilateral PVN pretreatment with the specific ANG II-AT 1 receptor antagonist losartan partially inhibited the i.c.v. ANG II-induced AVP release. We conclude: 1) Beta 1 and beta 2 adrenoceptors in the PVN exert an inhibitory action on basal AVP secretion. 2) ANG II can release AVP by directly stimulating its ANG II-AT 1 receptors in the PVN. 3) PVN mediated AVP release in response to periventricular ANG II-AT 1 receptor stimulation is at least partially effected through ANG II-AT 1 receptors in the PVN impinging on alpha adrenergic terminals.


Limitation of myocardial injury with the potassium channel opener cromakalim and the nonvasoactive analog U-89,232: vascular vs. cardiac actions in vitro and in vivo. Cromakalim has been shown to have anti-ischemic properties, but it also produces profound hypotension upon systemic administration. We hypothesized that U-89,232, a cromakalim analog, would reduce infarct size in an ischemia-reperfusion injury model without hemodynamic alteration. Twenty-four anesthetized, open chest New Zealand White rabbits were instrumented for occlusion of a marginal branch of the left coronary artery. All animals were subjected to coronary artery occlusion (30 min) and reperfusion (2 hr). Study animals received either cromakalim (20 micrograms/kg, i.v.) or U-89,232 (20 micrograms/kg, i.v.), which was given as a pretreatment 30 min before occlusion. Control animals (n = 10) received vehicle (10% dimethyl sulfoxide). At termination of the experiment, the necrotic area and the area at risk were determined with tetrazolium and India ink staining, and infarct size was calculated using planimetry. Treatment with cromakalim produced profound hypotension (greater than 30% decrease in mean arterial pressure), whereas U-89,232 had no such hemodynamic effect. With comparable areas at risk, infarct size (as a percent of risk area) in the control animals was 46.8 +/- 3.4%. Treatment with cromakalim or U-89,232 reduced infarct size to 33.1 +/- 4.4 and 24.4 +/- 4.0%, respectively (P < .05, both compared to control). In vitro studies demonstrate that although both of these compounds shorten the duration of the cardiac action potential, only cromakalim is active in vascular smooth muscle. We conclude that U-89,232 exhibits myoprotection without hypotension, and that its mechanism of action is most likely due to ability to affect cardiac electrophysiology.


Allosteric interactions between the binding sites of receptor agonists and guanine nucleotides: a comparative study of the 5-hydroxytryptamine1A and adenosine A1 receptor systems in rat hippocampal membranes. The ternary complex formed between agonist, receptor and guanine nucleotide binding protein and its destabilization by guanine nucleotides (GN) was utilized to study early events in signal transduction, by characterizing the allosteric interactions between agonist and GN binding to the receptor/guanine nucleotide binding protein, G complex for adenosine A1 and 5-hydroxytryptamine1A receptors. The functional interaction between the ternary complex and GTP was examined by assaying adenylyl cyclase activity. Binding of a full adenosine A1 agonist ([3H]-R-(-)-N6-(2-phenylisopropyl)adenosine), and a full [(+-)-[3H]-8-hydroxydipropylaminotetralin] and partial ([3H]-8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8- azaspirol[4.5]-decane-7,9-dione) 5-hydroxytryptamine1A agonist was examined in relation to the binding of GN. The amount of ternary complex formed depended upon receptor type and drug relative efficacy. The ratio between the drug's EC50 value (adenylyl cyclase) and dissociation constant (binding) was also receptor and drug relative efficacy dependent. 5'-Guanylylimidodiphosphate (100 microM) caused an approximately 50% decrease in the Bmax for all drugs without affecting Kd values. 5'-Guanylylimidodiphosphate and guanosine 5'-O-(3-thiotriphosphate) attenuated [3H]-agonist binding in a concentration-dependent and saturable manner, with IC50 values increased 2- to 6-fold with increasing receptor occupancy. IC50 values were approximately one-tenth lower at the 5-hydroxytryptamine1A receptor than adenosine A1 receptor; similar values were obtained for inhibition of (+-)-[3H]-8-hydroxydipropylaminotetralin and [3H]-8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8- azaspirol[4.5]-decane-7,9-dione binding, suggesting an independence of agonist efficacy. We propose that the stabilization of the ternary complex by hormone binding, measured by Bmax values, is related to drug-relative efficacy, thus the amount of ternary complex available for destabilization by GN is greater for the more efficacious agonist. This is translated into greater relative efficacy observed in the maximal inhibition of adenylyl cyclase.


Clearance and diffusion of locally applied dopamine in normal and 6-hydroxydopamine-lesioned rat striatum. Studies of dopamine (DA) clearance and diffusion were carried out after unilateral destruction of striatal DA afferents using intraparenchymal injection of 6-hydroxydopamine (6-OHDA). Rats were screened for the extent of DA depletion by testing for contralateral rotations induced by 0.05 mg/kg of apomorphine. Exogenous DA clearance and diffusion were determined using rapid (5 Hz) in vivo chronoamperometry with Nafion-coated carbon-fiber electrodes. Local applications of DA by pressure ejection from micropipettes into the lesioned vs. nonlesioned striata, with dose adjusted to elicit roughly equivalent amplitudes of electroactive signals, manifested a much prolonged clearance time on the lesioned side. A second protocol involved administration of equal volumes (amounts) of DA into lesioned vs. nonlesioned striata. In such cases, a volume of DA which manifested no detectable signal on the intact side produced a pronounced electrochemical signal on the lesioned side. When nomifensine, a high-affinity DA uptake inhibitor, was locally applied into the intact striatum, a subsequent ejection of DA produced a much larger signal than when the same volume was given before nomifensine. Very little or no effects of nomifensine were seen in 6-OHDA-lesioned striata. Taken together, these data indicate a much prolonged clearance time of DA in 6-OHDA-denervated striata. Moreover, the data also suggest that the primary mechanism underlying this effect is the loss of high affinity neuronal uptake. Thus, such changes in DA clearance may help account for some of the well documented compensatory phenomena which occur after DA-depleting lesions in animals and in man.


Differential effects of diazoxide, cromakalim and pinacidil on adrenergic neurotransmission and 86Rb+ efflux in rat brain cortical slices. The effects of diazoxide, cromakalim and pinacidil on depolarization-evoked tritium overflow from the rat brain cortical slices preloaded with [3H]noradrenaline were studied. Diazoxide inhibited both transmural nerve stimulation (TNS)- and 25 mM K(+)-evoked tritium overflows more potently than cromakalim. Diazoxide effects were only partially antagonized and cromakalim ones were totally reversed by 1 microM glibenclamide. Diazoxide, but not cromakalim, reduced the 45 mM K(+)-evoked tritium overflow, which was not antagonized by glibenclamide. Both diazoxide and cromakalim stimulated 86Rb+ efflux to a similar extent, the effects being completely abolished by glibenclamide. Glibenclamide (> or = 3 microM) by itself enhanced the TNS-evoked tritium overflow. Pinacidil increased both TNS- and K+ (25 and 45 mM)-evoked tritium overflows with little effect on 86Rb+ efflux. Pinacidil-induced increase in the TNS-evoked tritium overflow was still observed in the presence of cocaine or hydrocortisone. Pinacidil failed to affect the inhibitory action of xylazine on the TNS-evoked tritium overflow, whereas phentolamine attenuated it. These results indicate that ATP-sensitive K+ channels are present in the adrenergic nerve endings of rat brain. These channels seem to be pharmacologically different from those reported for vascular smooth muscles and pancreatic beta-cells.


The novel natural product YM-26567-1 [(+)-trans-4-(3-dodecanoyl-2,4,6- trihydroxyphenyl)-7-hydroxy-2-(4-hydroxyphenyl)chroman]: a competitive inhibitor of group II phospholipase A2. (+)-trans-4-(3-dodecanoyl-2,4,6-trihydroxyphenyl)-7-hydroxy-2-(4- hydroxyphenyl)chroman (YM-26567-1), a novel natural product isolated from the fruit of Horsfieldia amygdaline, dose-dependently inhibited group II phospholipase A2 (PLA2) prepared from rabbit platelet with an IC50 value of 6.7 microM (4.6-9.6 microM, n = 4). In contrast to irreversible PLA2 inhibitors such as manoalide and p-bromophenacyl bromide, the PLA2 inhibition of YM-26567-1 was independent of preincubation time. Lineweaver-Burk analysis revealed that YM-26567-1 behaved as a competitive inhibitor of rabbit platelet PLA2 with a Ki value of 1.6 +/- 0.3 microM (n = 5). Although YM-26567-1 also competitively inhibited group I PLA2 derived from porcine pancreas, the Ki value was approximately 10-fold greater for porcine pancreas than for rabbit platelet PLA2. In vivo, topical application of YM-26567-1 to the mouse ear inhibited 12-O-tetradecanoylphorbol-13-acetate (1 micrograms/ear)-induced mouse ear edema in a dose-dependent manner with a 50% effective dose of 28 micrograms/ear (13-63 micrograms/ear, n = 10/dose), but did not improve arachidonic acid (4 mg/ear)-induced mouse ear edema at 1 mg/ear. These results suggest that YM-26567-1 is a competitive PLA2 inhibitor showing a higher affinity for group II than group I PLA2, and that it may act as a potent anti-inflammatory compound through its direct inhibition of PLA2.


Whole body and brain distribution of [3H]cyclic [D-Pen2,D-Pen5] enkephalin after intraperitoneal, intravenous, oral and subcutaneous administration. The route of administration of a given drug can have a significant influence upon whole body distribution. The present study examined whole body distribution of the delta opioid receptor-selective peptide [3H]DPDPE in male CD1 mice after administration by several routes. Additionally, we describe regional brain distribution of [3H]DPDPE after i.v. administration with and without pretreatment with naloxone or the selective delta receptor antagonist naltrindole. Finally, characterization of the inherent enzymatic stability of DPDPE was also examined. Intravenous administration results in a significantly large amount of [3H]DPDPE in the small intestine and flush at 15 and 30 min postadministration, suggesting rapid biliary excretion. The highest level in the brain after i.v. administration occurred at 60 min (0.08%). After i.p. and s.c. administration, large amounts of [3H]DPDPE were found in the small intestine and flush, but not until 60 min postadministration, suggesting a slower rate of absorption from the site of administration. The i.p. and s.c. groups' brain levels peaked at 120 min (0.07 and 0.09%, respectively). The highest levels in the brain after p.o. administration were seen at 240 min (0.03%). Examination of regional brain distribution data showed no significant difference in the levels of [3H]DPDPE between brain regions at any time point studied. However, naloxone pretreatment resulted in significant reductions of [3H]DPDPE in all brain regions at 5 and 10 min. Naltrindole pretreatment resulted in significant reductions in the frontal cortex and striatum at 5 and/or 10 min postadministration, but had no effect on [3H]DPDPE levels in cerebellum, hippocampus or brain stem.(ABSTRACT TRUNCATED AT 250 WORDS)


Pardaxin induces exocytosis in bovine adrenal medullary chromaffin cells independent of calcium. Pardaxin, an excitatory neurotoxin, is a new tool for studying the machinery of neurotransmitter secretion. At noncytotoxic concentrations (< 1 x 10(-5) M), pardaxin stimulated exocytosis, as assessed by the concomitant release of catecholamines, ATP and dopamine-beta-hydroxylase from bovine adrenal medullary chromaffin cells in the presence or absence of extracellular calcium. At higher concentrations (> 2 x 10(-5) M), pardaxin was increasingly cytotoxic, as inferred from trypan blue uptake, release of lactate dehydrogenase and 51Cr (ED50 = 100 microM). The role of intracellular calcium ([Ca++]i) homeostasis in pardaxin action was investigated by using the fluorescent calcium indicator Fura-2. In the presence of extracellular calcium, addition of noncytotoxic concentrations of pardaxin yielded a steady, concentration-dependent rise in [Ca++]i (ED50 = 1 microM). Depolarization of chromaffin cells by high K+ reduced pardaxin binding and abolished the pardaxin-evoked rise in [Ca++]i. In the absence of extracellular calcium, pardaxin failed to elicit an elevation of [Ca++]i. These data suggest that, in the presence of extracellular calcium, pardaxin might cause elevations in [Ca++]i and neurotransmitter release, concomitant with inducing transmembranal Ca++ influx. However, the complex concentration dependence of [Ca++]i and the fact that pardaxin stimulated secretion without a rise of [Ca++]i suggest that the toxin, in addition to being a pore-forming molecule, might directly affect exocytosis in a Ca(++)-independent way. In proposing a pharmacological working model, we hypothesize that pardaxin might present a molecular structure which mimics an essential step in the endogenous docking mechanism between secretory granules and the plasma membrane.


Increase of the extracellular magnesium concentration reduces cardiac glycoside toxicity in the human myocardium. We studied the influence of magnesium on contractility and on force-frequency-relationship as well as on the positive inotropic and toxic effects of ouabain (OUA) on electrically driven human right auricular trabeculae. Radioligand binding experiments were performed with myocardial tissue from nonfailing and from terminally failing patients. Magnesium produced a concentration-dependent negative inotropic effect (P < .05). In contrast, OUA (0.03-0.1 mumol/l) concentration-dependently increased isometric force of contraction. The maximal positive inotropic effect of OUA (5.9 +/- 0.9 mN; 1 mmol/l of magnesium) was unchanged after increasing magnesium from 1 to 2 mmol/l (5.8 +/- 0.9 mN). OUA was as effective as Ca++ (15 mmol/l; 6.7 +/- 0.5 mN). OUA (0.05 and 0.03 mumol/l) exerted toxic effects after 2 hr and 0.08 or 0.1 mumol/l of OUA after 30 min, respectively. Time until toxic effects occurred after OUA (0.1 mumol/l) was significantly longer with 2 mmol/l of magnesium compared to 1 mmol/l of magnesium. In right auricular trabeculae, the force of contraction increased with increasing frequency (0.5-1 .5 Hz) of stimulation. The force-frequency-relationship becomes negative after elevation of extracellular Ca++ (2.4 mmol/l of Ca++) (2 Hz: 92 +/- 5.5% basal). Magnesium restored the force-frequency-relationship in the presence of enhanced Ca++ concentration (2.4 mmol/l of Ca++; 2 mmol/l of Mg++; 2 Hz: 145 +/- 16.9% basal). The receptor-density and affinity measured by [3H]OUA binding was not different in nonfailing and failing myocardium. Magnesium increased concentration-dependently the affinity of [3H]OUA to its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Positive inotropic effects of RP 62719, a new pure class III antiarrhythmic agent, on guinea pig myocardium. The mechanical effects of RP 62719 [(-)1-[-2-(3,4-dihydro-2H-1- benzopyran-4-yl)ethyl]-4-(3,4-dimethoxyphenyl)-piperidine] were tested in vitro on guinea pig left ventricular papillary muscle. RP 62719 is a novel pure class III antiarrhythmic agent known to prolong the cardiac action potential duration by selectively blocking the inward rectifying K+ current. Mechanical parameters were determined from contraction and relaxation phases under isotonic and isometric conditions. At a concentration of 0.02 microM, RP 62719 did not produce significant effects on inotropy or lusitropy. At 0.2 and 2 microM, the drug improved contraction under both heavy and low loading conditions, as evidenced by a 30% increase in maximum unloaded shortening velocity (Vmax, P < .001), peak amplitude of shortening (delta L, P < .001), peak isometric active force normalized per cross-sectional area (AF/s, P < .001) and positive peak of the force derivative per mm2 (+dF/s, P < .001). At the same concentrations, positive lusitropic effects were evidenced by an increase in maximum lengthening velocity (maxVr) and negative peak of force derivative per mm2 (-dF/s, P < .001). At a higher concentration (20 microM), effects of RP 62719 on inotropy and lusitropy were less marked, thus accounting for the bell-shaped form of the dose-response curve. An increase in the extracellular Ca++ concentration from 2.5 to 3.75 mM improved inotropy to a similar extent (+30-50%) as did 2 microM RP 62719. However, lusitropy and mechanical coupling between contraction and relaxation were not modified in the same proportion under RP 62719 and under 3.75 mM Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)


Quantitative autoradiography of muscarinic cholinergic receptor binding in the rat brain: distinction of receptor subtypes in antagonist competition assays. Prior studies have suggested the presence of muscarinic acetylcholine receptor (MAChR) subtypes within the mammalian central nervous system on the bases of functional ligand binding or molecular biologic evidence. Autoradiographic differentiation of MAChR subtypes in ligand binding assays has previously relied on the use of agonists, results of homogenate binding assays to determine relative subtype binding affinities or both. In the present study, the binding of [3H]scopolamine to intact slide-mounted tissue sections is characterized. The rapid binding kinetics of the ligand permit autoradiographic saturation experiments. Autoradiographic competition assays utilizing [3H]scopolamine and the unlabeled subtype-selective ligands pirenzepine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5,11,-dihydro-6H-pyr ido (2,3-b)(1,4)benzodiazepin-6-on (AF-DX 116) and 4-diphenylacetoxy-N-methylpiperidine methiodide reveal evidence for the presence of multiple MAChR subtypes distributed heterogeneously throughout the brain. High-affinity pirenzepine MAChR (putative M1 subtype) predominate in the telencephalon. High-affinity AF-DX 116 MAChR (putative M2 subtype) are widely distributed, but are quantitatively minor populations, with the exception of motor cranial nerve nuclei and the basal pons, where they represent the dominant MAChR fractions. Evidence for relative enrichment of M3 receptors was obtained in the thalamus, the superficial layer of the superior colliculus, the periqueductal region, the substantia nigra pars reticulata and the pons. The autoradiographic assays developed in this work may assist in defining altered receptor populations arising in pathologic conditions or resulting from drug therapy.


[125I]-labeled forskolin analogs which discriminate adenylyl cyclase and a glucose transporter: pharmacological characterization and localization of binding sites in rat brain by in vitro receptor autoradiography. Aminoalkylcarbamate derivatives of forskolin have been synthesized at the 6- and 7-hydroxyl positions which have different selectivity for adenylyl cyclase and a glucose transporter, respectively. They were radioiodinated using the Bolton-Hunter reagent to yield [125I]-2-[3-(4-hydroxy-3-iodophenyl)propanamido]-N-ethyl-6- (aminocarbonyl)forskolin ([125I]6-IHPP-Fsk) and [125I]-2-[3-(4-hydroxy-3-iodophenyl)(propanamidol]-N-ethyl-7- (aminocarbonyl)-7-desacetylforskolin ([125I]7-IHPP-Fsk) and tested as autoradiographic probes for adenylyl cyclase and a glucose transporter. In slide-mounted rat brain sections [125I]6-IHPP-Fsk binding was potently inhibited by 1 microM 6-HPP-Fsk (95%) but unaffected by 500 mM D-glucose. In contrast, [125I]7-IHPP-Fsk was only partially inhibited by 1 microM 6-HPP-Fsk (37%), but residual [125I]7-IHPP-Fsk binding was further inhibited 56% by 500 mM D-glucose. These data suggest that while [125I]6-IHPP-Fsk binds exclusively to adenylyl cyclase, a significant fraction of [125I]7-IHPP-Fsk is binding to a glucose transporter in brain. Autoradiographic patterns of [125I]6-IHPP-Fsk and glucose-sensitive [125I]7-IHPP-Fsk binding were different. [125I]6-IHPP-Fsk binding was heterogeneously distributed and resembled [3H] forskolin binding. Highest densities of binding sites were noted in olfactory tubercle, caudate putamen, nucleus accumbens, pyramidal and granule cell layers of hippocampus, molecular layer of cerebellum and substantia nigra. In contrast, of glucose-sensitive [125I]7-IHPP-Fsk, binding appeared more homogeneous and similar to [3H]cytochalasin B, a compound which inhibits glucose transport. Highest densities of binding were noted in caudate putamen, nucleus accumbens, cerebral cortex and molecular layer of cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)


Baroreflex control of heart rate in rats with heart failure after myocardial infarction: effects of captopril. Activation of the renin-angiotensin and sympathetic nervous systems in heart failure may result in altered baroreflex control of heart rate. To determine the specific effects of treatment with captopril on baroreceptor dysfunction in heart failure, baroreflex control of heart rate was measured in conscious rats with heart failure 6 weeks after ligation of the left coronary artery. Plasma norepinephrine was measured as a reflection of sympathetic nervous system activity. After bolus injections of phenylephrine (2-50 micrograms/kg) and nitroprusside (2-50 micrograms/kg), the arterial baroreflex was analyzed by fitting percentage of mean arterial pressure changes and heart rate changes to a logistic regression function. There were no differences in baroreflex function between normal and sham-operated rats. Plasma norepinephrine was increased (P < .05) in the heart-failure rats and did not change with captopril treatment. In untreated rats, heart failure increased (P < .05) the centering point by 900%, threshold by 243% and saturation by 89%, whereas decreasing (P < .05) the operational point by 73%. There was a decrease (P < .05) in the nitroprusside-related gain and an increase (P < .05) in phenylephrine-related gain, but the overall baroreflex gain was not changed. In heart-failure rats, captopril increased (P < .05) threshold, saturation and centering point and decreased (P < .05) operational point and nitroprusside- and phenylephrine-related gain abnormalities. The increase in operational point and decreases in threshold, saturation, centering point and phenylephrine-related gain were the results of a specific interactive effect of captopril in heart failure (P = .0033, .0176, .0509, .0217 and .0567, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Olfactory toxicity of beta,beta'-iminodipropionitrile in the rat. Following a pilot study which revealed olfactory epithelial degeneration induced by beta,beta'-iminodipropionitrile (IDPN), dose-response and time-course analyses were undertaken to further characterize the effects of IDPN on the olfactory system. Male rats were sacrificed at multiple time points ranging from 24 hr after a single dose to 56 days after three consecutive daily doses of IDPN (0-400 mg/kg i.p.). Nasal cavities were fixed, decalcified and embedded in paraffin; 5 microns sections were stained with hematoxylin and eosin, middle neurofilament protein antibody or olfactory marker protein antiserum. Olfactory bulbs were removed for slot blot analyses of glial fibrillary acidic protein, synapsin I and p38. Another group of rats was treated with saline or IDPN and perfused 6 hr or 1, 2, 3, 7, 14 or 28 days after the last dose. Olfactory bulb axonal degeneration was visualized using a modified Gallyas technique. Twenty-four hours after treatment with 200 or 400 mg/kg IDPN, there was severe, highly site-specific mucosal degeneration in the dorsal-medial nasal cavity; regeneration was incomplete 8 weeks later. IDPN increased olfactory bulb glial fibrillary acidic protein, peaking 7 days after three daily 400 mg/kg doses, and remaining significantly elevated 8 weeks after treatment. Olfactory bulbs contained substantial silver deposition in afferent axon bundles in the glomerular layer, beginning 24 hr after the first dose and persisting for 14 days after dosing. Although only a portion of the olfactory epithelium was damaged by IDPN, all axon bundles entering the olfactory bulb were involved, suggesting the lack of a clear topographic arrangement of sensory endings in the olfactory bulb.


Determinants of the in vitro interaction of polyaspartic acid and aminoglycoside antibiotics. The in vitro interaction of polyaspartic acid (PAA) with aminoglycosides was evaluated using double diffusion in agar, dialysis chambers and changes in the optical density of test solutions. The results document a reversible, presumably electrostatic interaction that is optimized at a pH of approximately 5.0, by the absence of proteins over 800 Da, and at a 20:1 or 10:1 molar ratio of aminoglycoside to PAA. The PAA-aminoglycoside complex lost antibacterial activity and the ability to inhibit pronase E enzymatic activity. These results allow generation of a hypothesis as to the mechanism whereby PAA prevents aminoglycoside experimental nephrotoxicity.


Regulation of cytochrome P450IA1 gene expression in a human intestinal cell line, Caco-2. The expression and inducibility of cytochrome P450IA1 isozyme was investigated in the human carcinoma cell line Caco-2 cultured between days 7 and 35 in the absence or the presence of various enzyme inducers such as 3-methylcholanthrene, beta-naphthoflavone (beta NF), dioxin, isosafrole, rifampycin, dexamethasone or phenobarbital. 7-Ethoxyresorufin O-deethylase activity (EROD) was maximal at day 25 when the differentiation of Caco-2 cells, characterized by the level of the brush border associated enzymes such as sucrase isomaltase and alkaline phosphatase, was higher. The inducibility of this enzyme activity was found to be maximal when cells were treated between days 7 and 10. After a 3-day treatment of Caco-2 cells with 50 microM beta NF, EROD achieved 36.6 +/- 14.6 pmol/min/mg compared to 2.5 +/- 1.1 pmol/min/mg in untreated cells. This enzyme activity appeared to be supported only by P450IA1 isozyme because: 1) EROD was quantitatively inhibited by alpha-naphthoflavone, a P450IA1-specific inhibitor; otherwise, phenacetin O-deethylation was completely abolished in the presence of alpha-naphthoflavone and not by furafylline, a P450IA2-specific inhibitor; 2) EROD was induced after treatment with 3-methylcholanthrene, beta NF and dioxin, which are P450IA1 inducers, but not by isosafrole, a P450IA2-specific inducer; 3) cytochrome P450IA1 apoprotein could be immunodetected by antibodies directed against rabbit cytochrome P450-LM6, orthologous to P450IA1, in polycyclic hydrocarbon-treated cells; 4) under the latter conditions, P450IA1 mRNA accumulation was specifically detected, but not P450IA2 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)


Angiotensin II influences the renal hemodynamic response to blockade of thromboxane A2 and prostaglandin H2 receptors. This study was designed to examine the influence of angiotensin II on the renal hemodynamic response to blockade of thromboxane A2 and prostaglandin H2 receptors with SQ29548 (2 mg/kg, i.v. bolus, plus 2 mg kg-1 hr-1 infusion) in anesthetized rats. In control rats without any pretreatment, SQ29548 did not change blood pressure, but increased renal blood flow from 7.0 +/- 0.4 to 7.7 +/- 0.4 ml min-1 g kidney weight-1 (P < .05) and decreased renal vascular resistance from 18.1 +/- 1.0 to 16.2 +/- 0.8 mm Hg/ml min-1 g kidney weight-1 (P < .05). In contrast, SQ29548 was without effect on renal blood flow or renal vascular resistance in rats pretreated with saralasin or captopril to block angiotensin II actions and formation, respectively. SQ29548 also increased renal blood flow and decreased renal vascular resistance in rats pretreated with captopril in which the plasma concentration of angiotensin II was fixed at elevated levels by concurrently infusing the peptide at doses ranging from 5 to 80 ng/min. In this experimental setting, the administration of SQ29548 reduced preglomerular vascular resistance selectively. Because, according to previous studies, SQ29548 does not interfere with the direct vasoconstrictor actions of angiotensin II, the renal vasodilatory effect of SQ29548 in rats with elevated plasma angiotensin II is attributable to interference with the operation of mechanisms of vasoconstriction mediated by activation of thromboxane A2-prostaglandin H2 receptors. We conclude that the status of the renin-angiotensin system is a determinant of the renal vasodilatory response to SQ29548.(ABSTRACT TRUNCATED AT 250 WORDS)


Vasoconstrictor and vasodilator effects of serotonin in the isolated rabbit kidney. The renal vascular effects of serotonin (5-HT) in vivo vary among preparations, which may reflect that this autacoid can modulate the vascular response to certain spasmogens. We investigated this phenomenon in the isolated rabbit kidney perfused under constant flow (5 ml/min) with Krebs-bicarbonate buffer. Dose-response experiments to 5-HT were conducted before and during an infusion of half-maximal effective doses of norepinephrine (NE), phenylephrine (PE) or endothelin-1 (ET-1). Each infusion was varied to raise the perfusion pressure (PP) from a baseline of 18-28 mm Hg to a level of 80-100 mm Hg. In the absence of infused NE, PE or ET-1, bolus injections of 5-HT had little or no effect. However, in the presence of NE, 5-HT injections of 20 pmol to 20 nmol caused a dose-dependent increase in PP; higher doses (0.4-4 mumol) of 5-HT caused a dose-dependent decrease in PP. Similar results with 5-HT were obtained in the presence of an infusion of PE. However, in the presence of ET-1, 5-HT injections elicited only a modest increase in PP, which was significantly less than the increase in the presence of NE; and they did not have the vasodilator effect on the isolated perfused kidney. Histamine injections only increased but did not decrease the PP in PE-precontracted renal vasculature. Infusion of 5-HT at 20 nmol/min potentiated the dose-dependent effect of NE on PP, but 5-HT infusion of 1 mumol/min attenuated the effect of NE on PP.(ABSTRACT TRUNCATED AT 250 WORDS)


NPC 15669 inhibits the reversed passive Arthus reaction in rats by blocking neutrophil recruitment. NPC 15669, N-carboxy-L-leucine,N-[(2,7-dimethylfluoren-9-yl)methyl]ester, has been shown to inhibit several inflammatory reactions that depend upon recruitment of neutrophils into the primary lesion. In the present study we examined the effects of NPC 15669 in the reversed passive Arthus reaction, an inflammatory reaction occurring in the skin of rats in response to intracutaneous injection of antigen followed by intravenous administration of antibody. In this model, immune complex formation activates complement, resulting in rapid recruitment of neutrophils to the site, which releases free radicals and proteases that damage capillaries, resulting in plasma leak. NPC 15669 inhibited the increased capillary permeability occurring in the reversed passive Arthus reaction in a dose-dependent manner, with an ED50 of 4 mg/kg. The agent similarly inhibited the recruitment of radiolabeled neutrophils as well as the accumulation of myeloperoxidase, a neutrophil marker. NPC 15669 in vitro inhibited the adherence of formyl-L-Met-L-Leu-L-Phe- or human recombinant C5a-activated neutrophils to endothelium, with IC50 values of 15 to 30 microM (ca. 4-9 micrograms/ml). Measurement of plasma NPC 15669 showed that at the ED50 dose, the average circulating concentration of drug was 5 micrograms/ml, consistent with the hypothesis that NPC 15669 exerts its anti-inflammatory effects by inhibiting neutrophil adherence to endothelium and recruitment into the inflammatory lesion.


Glomerular hemodynamics of the clipped kidney: effects of captopril and diltiazem. Glomerular hemodynamics of the clipped kidney in two kidney-one clip Goldblatt-hypertensive rats were studied by micropuncture techniques after administration of either captopril (20 mg/kg of b.wt./day p.o.) or diltiazem (3 mg/kg of b.wt./hr i.v.). Both drugs decreased mean arterial and poststenotic renal arterial pressure to comparable levels. Total kidney glomerular filtration rate was not significantly different in the three groups (0.78 +/- 0.09 in hypertensive controls, 0.62 +/- 0.11 after captopril and 0.74 +/- 0.07 ml/min/g of kidney weight after diltiazem). Glomerular capillary pressure fell significantly in both treated groups but was lower in captopril-treated animals (60 +/- 2 in hypertensive controls, 45 +/- 2 after captopril and 53 +/- 1 mm Hg after diltiazem, both P < .05 vs. hypertensive controls). Afferent arteriolar resistance was lowered in both treatment groups to the same extent, whereas only captopril decreased efferent arteriolar resistance. Single nephron filtration rate of cortical nephrons was not altered significantly by captopril due to a 96% increase in the ultrafiltration coefficient and a 58% rise in glomerular plasma flow, whereas diltiazem increased ultrafiltration coefficient by 62% but did not affect glomerular plasma flow. These studies indicate that, despite of decreased effective filtration pressure, glomerular filtration rate is well preserved after converting enzyme inhibition. Captopril and diltiazem have different effects on glomerular hemodynamics within the stenosed kidney.


Possible involvement of hyperinsulinemia and adrenergic activation in the pathogenesis of indomethacin-induced antral ulcers in nonfasted hamsters and refed rats. Subcutaneous doses of indomethacin (IND) produced mainly antral ulcers in refed rats, almost exclusively corpus erosions in fasted rats, but no antral or corpus mucosal insult in nonfasted rats. In contrast, IND-treatment in nonfasted hamsters caused antral ulcers but minimal corpus erosions. Mild and severe hyperinsulinemia were observed in refed rats and nonfasted hamsters, respectively. The hyperinsulinemia and antral ulcers in the animals were significantly attenuated by a pretreatment (80 mg/kg, i.v., in rats, 60 mg/kg, i.p., in hamsters) of streptozotocin. Furthermore, the antral ulcers were dose-dependently attenuated by prazosin, an adrenergic alpha-1 antagonist, and yohimbine, an adrenergic alpha-2 antagonist, but not by propranolol, an adrenergic beta antagonist. The large doses of atropine (3.2-10 mg/kg, p.o.) also attenuated the ulcers. The histamine H2 receptor antagonist, famotidine (1-10 mg/kg, p.o.), significantly attenuated the antral ulcers in nonfasted hamsters, but not in refed rats. The latter two drugs exhibited greater inhibition of the corpus erosions in fasted rats compared with the antral ulcers in refed rats and nonfasted hamsters. The corpus erosions were more extensive after streptozotocin treatment (80 mg/kg, i.v.) and were aggravated significantly by yohimbine, whereas they were hardly affected by prazosin or propranolol. It is thus concluded that hyperinsulinemia and adrenergic activation are commonly involved in the etiology of the IND-induced antral ulcers in refed rats and nonfasted hamsters. Vagal activity, which plays a crucial role in the etiology of the corpus erosions in fasted rats, may play a part in the antral ulcer formation in nonfasted hamsters, but not in refed rats.


Effects of Losartan, a nonpeptide angiotensin II receptor antagonist, on norepinephrine overflow and antidiuresis induced by stimulation of renal nerves in anesthetized dogs. We examined the role of endogenous angiotensin II on renal noradrenergic neurotransmission in anesthetized dogs, using Losartan, a nonpeptide angiotensin II receptor (angiotensin subtype 1) antagonist. The renal nerve stimulation caused a frequency-dependent increase in renal norepinephrine secretion rate. The low frequency renal nerve stimulation (0.5-2.0 Hz) significantly decreased urine flow and urinary excretion of sodium, without affecting renal hemodynamics. The high frequency renal nerve stimulation (2.5-5.0 Hz) produced a more potent antidiuresis and a renal vasoconstriction that resulted in reductions of renal blood flow and glomerular filtration rate. Intrarenal arterial infusion of Losartan (10 and 100 micrograms/kg/min) did not affect the basal levels of norepinephrine secretion rate, although increased urine formation with some renal vasodilation were observed during infusion of the drug. The administration of Losartan had an inhibitory action on the decreased urine formation, renal vasoconstriction and enhanced norepinephrine secretion rate, in response to renal nerve stimulation. Based on these findings, we suggest that endogenous angiotensin II seems to regulate renal noradrenergic neurotransmission by facilitating norepinephrine release, through the prejunctional angiotensin subtype 1 receptor.


Transport of glutathione at blood-brain barrier of the rat: inhibition by glutathione analogs and age-dependence. We showed previously that glutathione (GSH) may cross the blood-brain barrier intact by a saturable low affinity transport process (Km approximately 6 mM) (Kannan et al., J. Clin. Invest. 85: 2009-2013, 1990). In the present report, breakdown and resynthesis of GSH as the mechanism of apparent GSH uptake were excluded further because > 87% of injected 35S-cysteine taken up at the blood-brain barrier remained unchanged with negligible incorporation into GSH. In an effort to characterize further this GSH transport system, we have studied the influence of a number of potential inhibitors on brain uptake index (BUI) of GSH in rats pretreated with a gamma-glutamyl transpeptidase inhibitor, acivicin. The BUIs of tracer 35S-GSH uptake in the presence or absence of 1 to 20 mM cysteine, glutathione disulfide, gamma-glutamylglutamate, gamma-glutamyl-p-nitroanilide and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid did not differ significantly from each other. However, S-alkyl glutathiones (hexyl and octyl), sulfobromophthalein-glutathione, glutathione monoethyl ester, probenecid (5 mM) and ophthalmic acid (10 mM) inhibited GSH uptake significantly. Inhibition of GSH uptake by sulfobromophthalein-glutathione and GSH-monoethyl ester was concentration-dependent with apparent Ki approximately 0.016 and 0.083 mM, respectively. There was a decline in GSH-BUI as a function of age in both acivicin and nonacivicin-pretreated rats during the growth and developmental period from 25 to 135 days of age (100-500 g b.wt.). The decrease in BUI with age was specific for GSH; cysteine uptake did not change and no difference in diffusible (H2O) and nondiffusible (sucrose) components was found in this age range.(ABSTRACT TRUNCATED AT 250 WORDS)


Targeted delivery of human recombinant superoxide dismutase by chemical modification with mono- and polysaccharide derivatives. Four types of superoxide dismutase (SOD) derivatives such as SOD-carboxymethyl dextran conjugate, SOD-diethylaminoethyl dextran conjugate, galactosylated SOD and mannosylated SOD were synthesized and their potential for selective targeting to organs or cells was evaluated in mice by pharmacokinetic analysis. All SOD derivatives retained 50 to 80% of the original enzymatic activity and were stable during incubation with mouse serum retaining enzymatic activity greater than 80% for 3 hr. After intravenous injection, native SOD was rapidly excreted into urine and no significant accumulation was observed in the organs except the kidney. SOD-carboxymethyl dextran conjugate gave a long plasma half-life because of impaired glomerular filtration and tissue interaction. By contrast, galactosylated-SOD and mannosylated-SOD were very rapidly eliminated from the circulation and taken up by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis. These uptake processes were nonlinear and hepatic uptake clearance decreased as the dose increased, although almost complete extraction was obtained at a dose of 0.1 mg/kg. Furthermore, the accumulation in kidney of both glycosylated SODs was drastically decreased due to reduced renal proximal tubular reabsorption and also enhanced hepatic clearance. SOD-diethylaminoethyl dextran conjugate also rapidly disappeared from plasma and distributed into liver, but its accumulation occurred due to electrostatic interaction and was nonspecific in cellular distribution. These results suggest the possibility of controlling the in vivo fate of SOD at a cellular level by chemical modification utilizing sugar moieties with varied physicochemical and/or biological characteristics.


Induced release of acetylcholine from guinea pig ileum longitudinal muscle-myenteric plexus by anatoxin-a. Anatoxin-a (ANTX), a nicotinic agonist, has been shown to induce contraction of guinea pig ileum, which was abrogated by the muscarinic antagonist atropine and the nicotinic antagonists tubocurarine and hexamethonium. We showed here that the ganglionic nicotinic antagonist mecamylamine was a better inhibitor of the contraction of ileum induced by ANTX. The sodium channel blocker tetrodotoxin also abolished ANTX-induced contraction. In contrast, alpha-bungarotoxin, the muscle type nicotinic receptor blocker, had no effect on ANTX-induced contraction of guinea pig ileum. Longitudinal muscle-myenteric plexus prepared from guinea pig ileum, labeled with [3H]choline and then incubated with ANTX was shown for the first time to release [3H]acetylcholine (ACh) in a dose-dependent manner. Pretreatment of longitudinal muscle-myenteric plexus with tubocurarine, hexamethonium or mecamylamine blocked ANTX-induced release of [3H]ACh. In contrast, atropine was without effect. Mecamylamine was the most potent antagonist. As observed in ileum contraction, tetrodotoxin completely and potently blocked the release of [3H]ACh induced by ANTX. Neither alpha-bungarotoxin nor the neuromuscular junction blockers conotoxin G1 or M1 could inhibit the [3H]ACh release. Taken together, these results suggested that ANTX activated nicotinic receptors on ganglionic interneurons to trigger a release of ACh, which next stimulated muscarinic receptors and induced ileum contraction.


Diversity of form in the amphibian papilla of Puerto Rican frogs. In modern frogs, the amphibian papilla exhibits a caudal extension whose shape, relative length, and proportion of hair cells vary markedly from species to species. Tuning in the caudal extension is organized tonotopically and evidently involves the tectorium. In terms of the proportion of amphibian-papillar hair cells in the caudal extension, we report more diversity among 8 species of a single genus (Eleutherodactylus) on a single island (Puerto Rico) than has been found so far among all of the (more than 50) other modern anurans examined for this feature from around the world. These 8 Puerto Rican species have overlapping habitat and conspicuous diversity in the male advertisement call. For 7 of the 8 species, we report that the call has transient spectral components in the frequency range of the amphibian papilla, and that the proportion of caudal extension hair cells and the frequency distribution of those components are correlated. Thus one might conclude that the selective pressures that led to diversity of calls among the 8 species also led to diversity in form of the amphibian papilla.


Output connections of a wind sensitive interneurone with motor neurones innervating flight steering muscles in the locust. The output connections of a bilaterally symmetrical pair of wind-sensitive interneurones (called A4I1) were determined in a non-flying locust (Schistocerca gregaria). Direct inputs from sensory neurones of specific prosternal and head hairs initiate spikes in these interneurones in the prothoracic ganglion. The interneurone with its axon in the right connective makes direct, excitatory connections with the two mesothoracic motor neurones innervating the pleuroaxillary (pleuroalar, M85) muscle of the right forewing, but not with the comparable motor neurones of the left forewing. The connections can evoke motor spikes. The interneurones also exert a powerful, but indirect effect on the homologous metathoracic pleuroaxillary motor neurones (muscle 114), and a weaker, indirect effect on subalar motor neurones of the hindwings. No connections or effects were found with other flight motor neurones, or motor neurones innervating hindleg muscles, including common inhibitor 1 which also innervates the pleuroaxillary muscle. One thoracic interneurone with its cell body in the right half of the mesothoracic ganglion and with its axon projecting ipsilaterally to the metathoracic ganglion receives a direct input from the right A4I1 interneurone. These restricted output connections suggest a role for the A4I1 interneurones in flight steering.


Structure and function of the cochlea in the African mole rat (Cryptomys hottentotus): evidence for a low frequency acoustic fovea. The cochlea of the mole rat Cryptomys hottentotus was investigated with physiological and anatomical methods. In order to reveal the place-frequency map of the cochlea, iontophoretic HRP-applications were made in the cochlear nucleus at physiologically characterized locations. Subsequent HRP-transport in auditory nerve fibres and labeling patterns of spiral ganglion cells within the cochlea were evaluated. A cochlear place-frequency map was constructed from 17 HRP-applications in the cochlear nucleus at positions where neurons had characteristic frequencies between 0.1 and 12.6 kHz. As in other mammals, high frequencies were found to be represented at the cochlear base, low frequencies at the cochlear apex. The place-frequency map had three distinct parts which were characterized by their different slopes. A clear over-representation of the frequencies between 0.6 and 1 kHz was revealed, in this frequency range the slope of the place-frequency map amounted to 5.3 mm/octave. As calculated from the regression analysis, below 0.6 kHz the slope of the cochlear place-frequency map amounted to 0.24 mm/octave, above 1 kHz to 0.9 mm/octave. As in other mammals width of the basilar membrane (BM) increased from the cochlear base towards the cochlear apex. Also in concordance with the findings in other mammals, BM-thickness decreased from the cochlear base to the apex. However, it was remarkable to find that there was no or little change in BM-width and thickness between 40 and 85% BM-length. It was also revealed that scala tympani was only 1/10th the size found in the rat or other mammals of similar body size.(ABSTRACT TRUNCATED AT 250 WORDS)


Temporal coding of pheromone pulses and trains in Manduca sexta. We investigated the ability of pheromone-sensitive olfactory receptors of male Manduca sexta to respond to 20-ms pulses of bombykal, the major component of the conspecific pheromonal blend. Isolated pulses of bombykal elicited a burst of activity which decreased exponentially with a time constant of 160-250 ms. Trains of pulses delivered at increasing frequencies (0.5-10 Hz) elicited temporally modulated responses at up to 3 Hz. Concentration of the stimulus (1, 10, 100 ng per odor source) had a marginal effect on the temporal resolution of the receptors. Within a train, the responses to individual pulses remained constant, except for 10-Hz trains (short-term adaptation). A dose-dependent decline of responsiveness was observed during experiments (long-term adaptation). Although individual neurons may not respond faithfully to each pulse of a train, the population of receptors sampled in this study appears to be capable of encoding the onset of odor pulses at frequencies of up to at least 3 Hz.


Spectral selectivity of FM-FM neurons in the auditory cortex of the echolocating bat, Myotis lucifugus. 1. Spectral sensitivity was examined in delay-sensitive neurons in the auditory cortex of the awake FM bat, Myotis lucifugus. FM stimuli sweeping 60 kHz downward in 4 ms were used as simulated pulse-echo pairs to measure delay-dependent responses. At each neuron's best delay, the pulse and/or echo were divided into 4 FM quarters (Ist, IInd, IIIrd, and IVth), each sweeping 15 kHz in 1 ms, and quarters essential for delay sensitivity were determined for both pulse and echo. 2. For the pulse, the IVth quarter was essential for delay sensitivity in the majority of neurons. For the echo, the essential quarter for most neurons was the IInd, IIIrd, or IVth. 3. Different quarters of the pulse and echo were essential for delay sensitivity in 68% of the neurons examined. 4. This study provides neurophysiological evidence linking both spectral and temporal processing in delay-sensitive neurons of Myotis. Since spectral cues can provide target-shape information, sensitivity to both spectral and temporal parameters in single neurons may endow these neurons in FM bats with the potential for target analysis other than echo-ranging.


Circadian nature of the photoperiodic clock in Japanese quail. The photoperiodic clock in quail (Coturnix coturnix japonica) is based upon a rhythm of photoinducibility (phi i) but the extent to which this rhythm is circadian remains unclear. Two types of experiment investigated this situation. In the first, gonadectomized quail were adapted to live in periods of darkness by training them on a schedule containing one short day and 3 days of darkness (SD/DD/DD/DD). They were then exposed to a single pulse of 6 or 10 h of light at different times across 3 days of darkness. The photoperiodic response, measured by the increase in LH secretion, showed clear rhythmicity, demonstrating unequivocally the circadian nature of phi i. The second set of experiments employed Nanda-Hamner cycles and varied the length of the photoperiod from 6 to 11 h. Responsiveness in a 36 h or a 60 h cycle was highly dependent upon the length of the photoperiod, something not predicted from theory. For instance, LD 6:30 was not photoperiodically inductive but LD 10:26 was clearly inductive. Close analysis of patterns of LH secretion indicated an unexpected delay before induction occurred and then a rapid rise to a stable level of induction. When LH was measured in every pulse under LD 10:26 there was no evidence that LH levels alternately increased and decreased. This is not consistent with the simplest interpretation of Nanda-Hamner experiments where alternate pulses of light are thought to "entrain" the rhythm or "induce" a photoperiodic response by coinciding with phi i. It is concluded that the quail's photoinducible rhythm is indeed based on a circadian rhythm but one that is only weakly self-sustaining.(ABSTRACT TRUNCATED AT 250 WORDS)


The functional role of GABA and glycine in monaural and binaural processing in the inferior colliculus of horseshoe bats. The functional role of GABA and glycine in monaural and binaural signal analysis was studied in single unit recordings from the central nucleus of the inferior colliculus (IC) of horseshoe bats (Rhinolophus rouxi) employing microiontophoresis of the putative neurotransmitters and their antagonists bicuculline and strychnine. Most neurons were inhibited by GABA (98%; N = 107) and glycine (92%; N = 118). Both neurotransmitters appear involved in several functional contexts, but to different degrees. Bicuculline-induced increases of discharge activity (99% of cells; N = 191) were accompanied by changes of temporal response patterns in 35% of neurons distributed throughout the IC. Strychnine enhanced activity in only 53% of neurons (N = 147); cells exhibiting response pattern changes were rare (9%) and confined to greater recording depths. In individual cells, the effects of both antagonists could markedly differ, suggesting a differential supply by GABAergic and glycinergic networks. Bicuculline changed the shape of the excitatory tuning curve by antagonizing lateral inhibition at neighboring frequencies and/or inhibition at high stimulation levels. Such effects were rarely observed with strychnine. Binaural response properties of single units were influenced either by antagonization of inhibition mediated by ipsilateral stimulation (bicuculline) or by changing the strength of the main excitatory input (bicuculline and strychnine).


The efficacy of a peptide-nucleic acid solution (Reticulose) for the treatment of hepatitis A and hepatitis B--a preliminary controlled human clinical trial. To complete this preliminary study 53 patients were chosen. All patients were screened, diagnosed for Hepatitis A or Hepatitis B and separated into treatment and control groups. All of the patients selected were in the early onset stage of their infection, and diagnosed for Hepatitis A or B by appropriate laboratory tests with special attention to Anti-HAV, IGM and HepB surface Antigen to carefully differentiate those with A from those with B. The treated patients received Reticulose for a 15 day period and the control patients received placebo injections of physiological saline. Reticulose patients with Hepatitis showed positive. 18 patients with Hepatitis B and 9 patients with Hepatitis A were treated with Reticulose, compared to 17 control patients with Hepatitis B and 9 control patients with Hepatitis A treated with placebo. Based upon laboratory findings of several parameters; Prothrombin times, Serum bilirubin, white blood cell (WBC count) and clinical observations; Reticulose treated patients appeared to show significant improvement. The bilirubin levels of 83% of patients with Hepatitis B, treated with Reticulose for 15 days were in normal range in 30 days. None of the control patients were within normal range after 30 days with placebo treatment. Of Hepatitis A patients treated with Reticulose, 100% showed normal bilirubin after 30 days. Of control patients with Hepatitis A, only 22% were in normal range after 30 days. The findings in this preliminary trial lead to the conclusion that Reticulose appears to significantly reduce the recovery time and return to normal for patients with an acute episode of Hepatitis A or B. Further study is indicated.


Dietary habits of technical and vocational students in Riyadh, Saudi Arabia--Part II. Eating between meals. Dietary habits of 452 young adult male students at 6 technical institutes and vocational training centres in Riyadh, Saudi Arabia were investigated as part of a nutritional assessment survey. The eating-between-meals habit was found to be common among the majority (72.3%) of those students. The association between this habit and age cohorts, marital status, accommodation status, self assessment of weight and exercising are discussed.


Road traffic injuries in Al-Ain City, United Arab Emirates. Road traffic injuries are the second major cause of death in the United Arab Emirates (UAE). A retrospective study for a 1 year period of all road traffic accidents and injuries was conducted in Jimi Hospital for the period 1 January to 31 December, 1990. A total of 1197 injuries were encountered at the Hospital Emergency Department. The majority of victims were males and under the age of 35 years. During the Ramadan (fasting month), the number of persons who were injured by traffic accidents was slightly higher than during other months. Excessive speed was closely related to traffic accidents and injuries. Most of the accidents and injuries occurred from 8.00 am to 2.00 pm. Head injury was the most frequently noted type of injury (40.5%), followed by limbs injury (22.1%) and chest, abdomen and pelvic injuries (14.7%). These findings suggest that there is a need to take a comprehensive approach on both excessive speed and driving habits.


Crohn's disease & aflatoxins.

Crohn's disease & aflatoxins. An investigation to examine the relationship between Crohn's disease and aflatoxins, a group of structurally related toxic and carcinogenic metabolites, was carried out on 24 patients. Extracts of serum and urine from the patients were assayed qualitatively by thin layer chromatography and the Aflatest method, and quantitatively by fluorimetry. There was evidence that some patients suffering from Crohn's Disease, together with some having coeliac disease and ulcerative colitis, did have varying amounts of aflatoxins in their serum and urine. The presence of aflatoxins may have been due to exposure to food containing these toxins or inability of the patient to excrete aflatoxins on account of some gastro-intestinal derangement. Only long-term investigation would establish the link between dietary history and the presence of aflatoxins in these patients.


The effect of selected health education schemes on knowledge and attitude of the Kanuri towards certain parasitic diseases. The effect of 3 types of intervention schemes on the scientific knowledge and attitude of Kanuri towards malaria, diarrhoea and dysentery and intestinal worms was assessed using the Likert scale response type statements. 1383 respondents in our identical Kanuri villages were used in the study. The effect of still pictures and card games (Goramari), drama songs, storytelling and discussions (Yerimari) and a combination of both (Shetimari) on knowledge and attitude was tested. Gotimari was used as the control. Quarterly assessment of changes in knowledge and attitude showed that Goramari was the least effective while Yerimari and Shetimari were the most effective. Women were more influenced than men by health education schemes which involve drama songs and stories while card games and still pictures had very little effect on them (women). The implication of these findings on disease management through health education is discussed.


Zimbabwe's success story in education and health: will it weather economic structural adjustment? The beginning of the 1980s saw the birth of Zimbabwe as a result of a protracted liberation war. It coincided with global interest in primary health care, the concept of universal primary school education and, unfortunately, moves towards economic stabilization and structural adjustment programmes. Economic structural adjustment was adopted by several sub Saharan African countries with dire consequences for the poor and vulnerable. Zimbabwe's commitment to social justice and to equitable distribution of resources demonstrated a practical move away from the culture of rhetoric so characteristic of many governments and non-governmental organisations and agencies. This commitment has been translated into impressive improvements in health and education. Current evidence shows that education has had a positive impact on health and related areas like contraceptive use, child mortality and the nutrition status of children. Conversely nutrition and health conditions among school children are important determinants of educational outcomes. Hitherto Zimbabwe's economy has been sufficiently strong to avoid excessive dependence on the International Monetary Fund, the World Bank and other foreign financial institutions. Unfortunately, however, the current economic recession together with economic structural adjustment programmes are beginning to have a negative impact on health and education. Will true synergism between health and education weather these structural problems? It seems that the people and government of Zimbabwe have the capacity and resolve to weather such a storm.


Building materials and environmental health: radon in Hong Kong. The origin and health effect of radon are first identified. In Hong Kong, the main contribution to the indoor radon concentration comes from building materials. The relatively high radium content in building materials used in Hong Kong explains the high indoor radon concentrations measured. Using risk analysis and established data, radon induced lung cancer deaths have been estimated and remedial measures have been recommended. The variations with age of building and type of ventilation are stated to be additional controlling factors. The possible effects of radon solubility in water are also explored.


Synthesis and structure-activity relationships of acetylcholinesterase inhibitors: 1-benzyl-4-(2-phthalimidoethyl)piperidine and related derivatives. Following the discovery of a new series of 1-benzyl-4-[2-(N-benzoyl-N-methylamino)ethyl]piperidine (2) derivatives with a potent anti-acetylcholinesterase (anti-AChE) activity, we extended the structure-activity relationships (SAR) to rigid analogues (4) and 1-benzyl-4-[2-(N-benzoyl-N-phenylamino)ethyl]piperidine derivatives (3). Introduction of a phenyl group on the nitrogen atom of the amide moieties resulted in enhanced activity. The rigid analogue containing isoindolone (9) was found to exhibit potent anti-AChE activity comparable to that of 2. Furthermore, replacement of the isoindolone with other heterobicyclic ring systems was examined. Among the compounds prepared in these series, 1-benzyl-4-[2-[4-(benzoylamino)phthalimido]ethyl]piperidine hydrochloride (19) (IC50 = 1.2 nM) is one of the most potent inhibitors of AChE. Compound 19 showed a definite selectivity to AChE over the BuChE (about 34700-fold) and, at dosages of 10-50 mg/kg, exerted a dose-dependent inhibitory effect on AChE in rat brain.


2-(N'-alkylidenehydrazino)adenosines: potent and selective coronary vasodilators. The reaction of aliphatic aldehydes and ketones with 2-hydrazinoadenosine under relatively mild conditions (at room temperature or in refluxing methanol) formed 2-(N'-alkylidenehydrazino)-adenosines, 5-22, in good yields. Two kinds of adenosine receptors regulate cardiac and coronary physiology. In supraventricular tissues an A1AR coupled to muscarinic K channels mediates the negative chronotropic, dromotropic, and inotropic actions of adenosine, and an inhibitory A1AR coupled to adenylate cyclase mediates the "antiadrenergic" action of adenosine. One or more kinds of A2 receptors mediate coronary vasodilation. Bioassays employing a guinea pig heart Langendorff preparation showed that 5-22 weakly retard impulse conduction through the AV node (negative dromotropic effect), but several analogues were very active coronary vasodilators. The coronary vasoactivity of the (n-alkylidene- and of the (isoalkylidenehydrazino)adenosines paralleled the length of the alkyl chain, the EC50s of the of the most active n-pentylidene (8) and isopentylidene (18) congeners being 1 nM. The EC50s of the cyclohexylmethylene (9), cyclohexylethylidene (10), and cyclohex-3-enylmethylene (12), analogues were likewise < 1 nM, but the cyclohex-1-enylmethylene congener 12 was 10 times less active than 9. The unselective adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (0.1 mM) raised the EC50s of the negative dromotropic effects of 8, 9, and 18 by 5-28-fold and the EC50s of coronary vasodilation of 22-90-fold. Catalytic reduction of 9 increased the hydrophobicity and changed the UV spectrum, suggesting reduction of the --CH = N-- bond. The product darkened on exposure to air and so was not characterized further. A new method for preparing 2',3',5'-tri-O-acetyl-2,6-dichloropurine riboside, a precursor in the synthesis of 2-hydrazinoadenosine, consists of the addition of tert-butyl nitrite to a mixture of 2',3',5'-tri-O-acetyl-6-chloroguanosine and CuCl in CHCl3 saturated with Cl2.


2-(N'-aralkylidenehydrazino)adenosines: potent and selective coronary vasodilators. This study aimed at the development of 2-(N'-aralkylidenehydrazino)adenosines as coronary vasodilators. The reaction of aromatic aldehydes or ketones with 2-hydrazinoadenosine in refluxing methanol formed the target compounds 2-27 as crystalline products in good yields. Two kinds of receptors mediate the actions of adenosine on the heart. Retardation of impulse conduction through the atrioventricular node, the negative dromotropic action, is an example of adenosine's action at an A1 receptor (A1AR) and coronary vasodilation reflects adenosine's action at an A2 receptor (A2AR). Accordingly, bioassays employing guinea pig heart Langendorff preparations assessed the selectivity of 2-27 as coronary vasodilators. Analogues 2-27 were weak negative dromotropic agents; the EC50 of the most active analogue, 2-[N'-(1-naphthylmethylene)hydrazino]-adenosine, 23, was 0.8 microM, several orders of magnitude less than many A1AR agonists. Some of the analogues were quite active coronary vasodilators; 2-(N'-benzylidenehydrazino)adenosine, 2, and several of its para-substituted derivatives, namely, the fluoro (7), methyl (13), methoxy (16), and tert-butylcarbonylethyl, 31, had EC50s for coronary vasodilation in the range 1.7-3.2 nM. The selectivity ratios, EC50 (negative dromotropic)/EC50 (coronary vasodilatory), of these five analogues ranged between 5100 (analogue 31) and 43,000 (analogue 2). Phenyl ring substitutions of other kinds or at other positions, replacement of the phenyl ring by other aryl or heteroaryl groups, or the replacement of the benzylic H by a methyl group lowered coronary vasoactivity significantly. The unselective adenosine receptor antagonist 8-(p-sulfophenyl)theophylline raised the EC50 of the negative dromotropic activities of 2, 16, and 2-[N'-(2-naphthylmethylene)hydrazino]adenosine, 24, by 3-, 18-, and 7-fold, and raised the EC50s of coronary vasoactivity by 11-, 3-, and 30-fold, respectively evidence that vasoactivity was receptor-mediated.


Dihydroxynitrobenzaldehydes and hydroxymethoxynitrobenzaldehydes: synthesis and biological activity as catechol-O-methyltransferase inhibitors. A series of nitro derivatives of dihydroxy- and hydroxymethoxybenzaldehyde was synthesized and tested as potential inhibitors of partially purified pig liver catechol-O-methyltransferase (COMT). All the dihydroxynitrobenzaldehydes prepared were potent inhibitors of COMT, but only one hydroxymethoxynitrobenzaldehyde (3-hydroxy-4-methoxy-5-nitrobenzaldehyde) showed activity as a COMT inhibitor. Although previously reported data showed that the presence of electron-withdrawing substituents at position 5 seemed to be very important for activity as COMT inhibitor, our results suggest that the requirement necessary to enhance the activity of the dihydroxyni-trobenzaldehyde derivatives toward COMT is the presence of the nitro group in a position ortho with respect to one hydroxyl group. The assayed compounds showed a reversible inhibition of COMT, which was mixed for all the dihydroxynitro derivatives but noncompetitive for 3-hydroxy-4-methoxy-5-nitrobenzaldehyde when pyrocatechol was the variable substrate and uncompetitive in all the inhibitors with respect to S-adenosyl-L-methionine.


Structure-activity relationships of imidazo[4,5-f]quinoline partial structures and analogs. Discovery of pyrazolo[3,4-f]quinoline derivatives as potent immunostimulants. Structure-activity studies have been carried out on a series of imidazo[4,5-f]quinoline derivatives reported to have potent in vivo immunostimulatory activity. This activity has been confirmed, and subtle structure-activity relationships have been uncovered which resulted in the identification of novel analogs (pyrazolo[3,4-f]quinoline derivatives, 7a,b) with potent in vivo effects in a mouse protection model. Regioisomeric pyrazolo[4,3-f]quinoline derivatives (6a,b) were shown to be inactive. Data are presented which support the notion that the in vivo activity is mediated by an immunostimulatory mechanism.


Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site. Hippocampal CA1 pyramidal cell neurons are sensitized over 30-fold to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid. This phenomenon has been termed the QUIS effect. In the present study several novel L-quisqualic acid analogues have been synthesized and tested for their interaction with the different components of the QUIS-effect system. Replacement of the oxadiazolidinedione ring of L-quisqualic acid with several other types of heterocyclic rings yielded the following quisqualic acid analogues: maleimide 2, N-methylmaleimide 3, N-(carboxymethyl)maleimide 4, succinimides 5A and 5B, and imidazolidinedione 6. None of these analogues were able to mimic the effects of L-quisqualic acid and sensitize hippocampal CA1 neurons to depolarization by L-AP4. Also, unlike L-serine O-sulfate, L-homocysteinesulfinic acid, or L-alpha-aminoadipic acid, none of the analogues were able to preblock or reverse the QUIS effect. However, when the IC50 values for inhibition of the CA1 synaptic field potential of analogues 2-6 were determined both before and after hippocampal slices were exposed to L-quisqualic acid, the IC50 values of analogues 3 and 4 were found to decrease more than 7-fold. Thus, these two compounds behave like L-AP4 rather than L-quisqualic acid in this system in that they exhibit increased potencies in slices that have been pretreated with L-quisqualic acid even though they cannot themselves induce this sensitization. Compounds 3 and 4, therefore, represent the first non-phosphorus-containing compounds to which hippocampal neurons become sensitized following exposure to L-quisqualic acid. No change in the IC50 values was observed for 5A or 5B. Analogues 2 and 6, on the other hand, displayed a high potency for inhibition of the evoked field potential even prior to treatment of the slices with L-quisqualic acid.


Novel spirosuccinimides with incorporated isoindolone and benzisothiazole 1,1-dioxide moieties as aldose reductase inhibitors and antihyperglycemic agents. Compounds from two novel series of spirosuccinimides were prepared. Analogs of series 2 possessed a spiro-fused isoindolone moiety while those of series 3 contained a spiro-fused benzisothiazole S,S-dioxide group. These compounds were evaluated as aldose reductase inhibitors (ARI) in vitro by their ability to inhibit glyceraldehyde reduction using a partially purified bovine lens aldose reductase preparation and in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. Many members from the isoindolone series 2, particularly those containing an isoindolone N-methyl moiety, showed good in vitro and in vivo potency. The most potent member, the 6-chloro analog 32, was resolved, and aldose reductase activity was found to reside almost exclusively in the (+)-enantiomer. Compound 32 was approximately equipotent in the sciatic nerve of the galactose-fed rat to other cyclic imide ARI's of similar in vitro activity, namely sorbinil and ADN-138 and also to tolrestat, an acetic acid-based ARI (ED50's 4-8 mg/kg). Compounds from both series, 2 and 3, were also found to lower plasma glucose levels of genetically obese db/db and ob/ob mice with potency similar to that of ciglitazone. However, members from these series failed to lower insulin levels of the ob/ob mouse at the doses tested.


NMR studies on YSPTSPSY: implications for the design of DNA bisintercalators. NMR (600 MHz) studies on YSPTSPSY (1), the heptad repeat unit of RNA polymerase II with an extra tyrosine added to the N-terminus, show that the peptide is partially structured in aqueous solution. Peptide 1 contains two overlapping SPXX sequences and has been reported to bind to DNA by bisintercalation (Suzuki, M. Nature 1990, 344, 562-565). In 90% H2O solution at pH 3.2, the major species, which is present in > 90%, contains Pro3 and Pro6 in the trans conformation. At low temperature (4 degrees C), NOE connectivities are consistent with the presence of beta-turn structures in equilibrium with unfolded forms of the peptide. A strong dNN connectivity between Thr4/Ser5, a d alpha N connectivity between Pro3/Thr4, and a medium-range NOE between Pro3 alpha and Ser5 NH indicate the presence of a beta-turn formed by (i) Ser2-Pro3-Thr4-Ser5. A strong dNN connectivity between Ser7/Tyr8 and weaker dN delta NOE connectivities between Ser2/Pro3 delta and Ser7/Pro6 delta were also detected. The solution conformation of the peptide appears to have a crucial role in determining the interaction of the peptide with DNA, given that only bisintercalation has been reported in DNA-binding studies on 1 (Suzuki, M. Nature 1990, 344, 562-565). On the basis of these results, the peptide unit--SPTSPS--(3) has considerable potential as a structured linker in the preparation of DNA bisintercalators of the general structure Xaa-SPTSPS-Zaa (2) with improved selectivity properties.


A novel orally active inhibitor of IL-1 generation: synthesis and structure-activity relationships of 3-(4-hydroxy-1-naphthalenyl)-2-propenoic acid derivatives. A new series of 3-(4-hydroxy-1-naphthalenyl)-2-propenoic acids was prepared and the inhibitory activities of its members on IL-1 generation were evaluated both by in vitro systems using human monocytes and/or rat exudated macrophages stimulated with LPS, and by an in vivo system using the rat CMC-LPS air-pouch model. Many compounds in this series were found to be potent inhibitors of IL-1 generation both in vitro and in vivo. Structure-activity relationships indicated that in the rat CMC-LPS air-pouch model by oral administration the (Z)-2-substituted propenoic acids with 3-alkoxy, 5-alkyl, and 4-hydroxy substituents on the naphthalene ring exhibit optimal inhibition. Among the compounds evaluated, (Z)-3-(5-ethyl-4-hydroxy-3-methoxy-1-naphthalenyl)-2-methyl-2-propeno ic acid (20a), which inhibited IL-1 generation from human monocytes with an IC50 value of 3.0 microM and had an IC50 value of 1.4 microM for rat exudated macrophages, showed the most potent inhibitory activity in the rat CMC-LPS model by oral administration. Compound 20a also showed antiinflammatory effects in animal models of inflammation.


Radiosynthesis, cerebral distribution, and binding of [125I]-1-(p-iodophenyl)-3-(1-adamantyl)guanidine, a ligand for sigma binding sites. An analog of 1,3-di-o-tolylguanidine (DTG), [125I]-labeled 1-(p-iodophenyl)-3-(1-adamantyl)guanidine (PIPAG), was synthesized as a potential ligand for cerebral sigma binding sites. Data from in vitro binding experiments and in vivo experiments on brain distribution suggested that PIPAG binds to sigma binding sites with high affinity (Kd in low nanomolar range) as determined by Scatchard analysis and relative potencies of sigma-specific drugs. Haloperidol had the highest potency to inhibit [125I]PIPAG binding. It was followed by DTG, BMY 14802, and (+)-N-allylnormetazocine. Compounds with high affinities for dopamine receptors (but low affinity for sigma binding sites), for opioid receptors, for nicotinic acetylcholine receptors, and for phencyclidine receptors were ineffective inhibitors of [125I]PIPAG binding.


Synthesis and biological evaluation of novel potent antagonists of the bombesin/gastrin releasing peptide receptor. This paper reports the synthesis and antagonist activity of 20 C-terminal analogues of gastrin releasing peptide (GRP). The ability of each analogue to inhibit bombesin (BN) stimulated amylase release from rat pancreatic acini was determined, and those showing antagonist activity were further evaluated for their ability to inhibit BN-stimulated [3H]thymidine uptake in serum-starved 3T3 cells. The assays also included two known peptide antagonists, C (Leu14,psi 13,14]BN) and H (N-pivaloyl-GRP20-25-(R)-2-methyl-4-nonylamide) as positive controls. On the basis of these assays we suggest that a des-Met27,Leu26-psi[CH2NHCOCH3]GRP C-terminal octapeptide imparts antagonist activity. The two most active compounds are peptides 14 ([D-Phe19,Leu26-psi(CH2NHCOCH3)]GRP19-26) and 18 ([D-Phe19,Gln20,Leu26-psi(CH2NHCOCH3)]GRP19++ +-26). In their ability to inhibit BN-stimulated [3H]thymidine uptake, the IC50 of peptides C, H, 14, and 18 were 43.2, 31.2, 2.7, and 32.5 nM, respectively. In conclusion, the novel C-terminal psi[CH2NHCOCH3] bond promises to be a useful peptide backbone modification for imparting antagonism in GRP/BN analogues.


Synthesis, configuration, and activity of isomeric 2-phenyl-2-(N-piperidinyl)bicyclo[3.1.0]hexanes at phencyclidine and sigma binding sites. The novel semirigid derivatives (+)-cis-1-[2-phenyl-2-bicyclo[3.1.0]hexyl]piperidine [(+)-8], its enantiomer (-)-8, and (+-)-trans-1-[2-phenyl-2-bicyclo[3.1.0]hexyl]piperidine [(+/-)-9] were synthesized as probes to investigate the mode of interaction of phencyclidine (PCP) with its binding site on the N-methyl-D-aspartate receptor complex. Each target compound was obtained in five steps starting from cyclopent-2-enone. (+)- and (-)-8 were obtained in greater than 98% optical purity through three recrystallizations from ethanol of the (S)-(+)- and (R)-(-)-mandelate salts of intermediate (+-)-cis-2-phenyl-2-bicyclo[3.1.0]hexylamine ([(+/-)-16]. Crystallization of the (R)-(-)-mandelate salt afforded (1R,2R,5S)-(-)-16, whereas the (S)-(+)-mandelate salt afforded (1S,2S,5R)-(+)-16; the absolute configuration was determined by single-crystal X-ray analysis of (-)-16.(R)-(-)-mandelate. Single-crystal X-ray analysis of (+/-)-9-picrate confirmed its trans configuration and provided conformational data. (+)- and (-)-8 and (+/-)-9 were examined for their ability to interact with PCP and sigma binding sites in vitro using [3H]TCP and [3H]pentazocine as radioligands. The binding was compared with that of PCP and contrasted with the rigid symmetrical phencyclidine derivatives cis- and trans-1-[3-phenyl-3-bicyclo[3.1.0]hexyl]piperidines (6 and 7). The results of the study indicated that the conformations of PCP represented by 6-9 are not optimal for potent interaction at either of these sites. Affinities ranged from 582 nM [(+/-)-9] to 29,000 nM [(+)-8] at PCP binding sites and from 1130 nM [(-)-8] to 16,300 nM (7) at sigma sites. In this assay, PCP exhibited affinities of 64.5 nM at PCP and 1090 nM at sigma sites. Qualitative correlation between the sigma and PCP binding data suggests some similarities between these binding sites. An axial phenyl and equatorial piperidine ring with the nitrogen lone pair of electrons antiperiplanar to the phenyl ring has been postulated as the receptor-active conformation of PCP-like ligands at the PCP binding site. Comparison of the binding data of 7-9 with that of the previously described methylcyclohexyl-PCP derivatives allowed its rationalization in terms of this model. It is likely that the lowered affinity in this bicyclo[3.1.0]hexane series is a consequence of nonoptimal geometry (pseudoequatorial phenyl or pseudoboat) for binding as opposed to the presence of steric bulk which proved deleterious in the methylcyclohexyl-PCP derivatives.


Synthesis and antiviral activity of deoxy analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent and selective anti-HIV-1 agents. The effect of substitution in the acyclic structure of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine (HEPT) on anti-HIV-1 activity was investigated by synthesizing a series of deoxy analogs and related compounds. Preparation of 1-[(2-alkyloxyethoxy)methyl]-6- (phenylthio)thymine (2-4) derivatives was carried out based on alkylation of HEPT with primary alkyl halides. Preparation of the 1-[(alkyloxy)methyl]-6-(phenylthio)thymine (26-31) and 1-[(alkyloxy)methyl]-6-(arylthio)-2-thiouracil (32-45) derivatives was carried out on the basis of LDA lithiation of 1-[(alkyloxy)-methyl]thymine (9-14) and 1-[(alkyloxy)methyl]-2-thiouracil (15-25) followed by reaction with diaryl disulfides. The oxidative hydrolysis of the 2-thiouracil derivatives gave 1-[(alkyloxy)methyl]-6-(arylthio)uracil derivatives (46-57). 1-Alkyl-6-(phenylthio)thymine (59-61) derivatives were prepared on the basis of alkylation of 6-(phenylthio)thymine (58). Methylation of the hydroxyl group of HEPT did not affect the anti-HIV-1 activity of HEPT. Substitution of the 1-(2-hydroxyethoxy)methyl group by ethyl, butyl, methoxymethyl, (propyloxy)methyl, and (butyloxy)-methyl groups somewhat improved the original anti-HIV-1 activity of HEPT. Substitution with ethoxymethyl and (benzyloxy)methyl groups further potentiated the activity [EC50: 1-(ethoxy-methyl)-6-(phenylthio)thymine (27), 0.33 microM; 1-[(benzyloxy)methyl]-6-(phenylthio)thymine (31), 0.088 microM]. When the 5-methyl group of 27 and 31 was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity was improved remarkably [EC50: 5-ethyl-1-(ethoxymethyl)-6-(phenylthio)-uracil (46), 0.019 microM; 5-ethyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (52), 0.0059 microM; 5-isopropyl-1-(ethoxymethyl)-6-(phenylthio)uracil (55), 0.012 microM; 5-isopropyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (56), 0.0027 microM]. Introduction of two m-methyl groups into the phenylthio ring also potentiated the activity.


Structural alterations in desferrioxamine compatible with iron clearance in animals. The design, synthesis, and biological evaluation of amideless desferrioxamine analogues are described. The design concept is predicated on the idea that a low molecular weight desferrioxamine analogue would represent a better pharmacophore from which to construct an orally effective or more efficient trihydroxamate than the parent chelator. The study demonstrates that (1) the monohydroxamate units of desferrioxamine must be linked to promote iron clearance, (2) the N-propanoyl-N-pentyl fragments of desferrioxamine can be replaced with smaller, e.g., C-5, methylene units without compromising the analogue's iron-clearing properties, and (3) a delicate balance exists between the molecule's iron-clearing efficiency and its lipophilicity.


Fluoroquinolones: relationships between structural variations, mammalian cell cytotoxicity, and antimicrobial activity. Fluoroquinolones are potent inhibitors of bacterial topoisomerase II (DNA gyrase). They can also inhibit eukaryotic topoisomerases, which could possibly lead to clastogenicity and/or cellular toxicity. Recent studies have demonstrated a correlation between mammalian cell cytotoxicity of the fluoroquinolones and the potential of these compounds to induce micronuclei, a genetic toxicity endpoint. In an effort to identify potent nontoxic quinolone antibacterials, we have examined the structural features of the fluoroquinolones associated with mammalian cell cytotoxicity. An investigation of a wide variety of substituents at the 1, 5, 7, and 8 positions of a quinolone nucleus was conducted. The results indicate that no one position has a controlling effect on the observed cytotoxicity. Instead, a combination of the various substituents contributes to the effects seen. Certain trends were apparent, such as the fact that compounds with pyrrolidines at the R-7 position were more cytotoxic than those with piperazines, and halogens at R-8 (X-position) were associated with more cytotoxicity relative to hydrogen. A general trend also existed between the cytotoxicity of the compounds and their Gram-positive antibacterial activity. A detailed comparison between the various groups and positional variations as they controlled the cytotoxicity and antibacterial activity is presented.


Dihydropyrimidine angiotensin II receptor antagonists. The discovery of the nonpeptide angiotensin II (AII) receptor antagonist losartan, previously called DuP 753, has stimulated considerable interest in the synthesis of novel analogs of this compound. Our efforts in this area have resulted in the discovery of dihydropyrimidines as potent AII receptor antagonists. The chemistry leading to this novel class of AII antagonists and their biological properties are reported in this publication. Structure-activity studies showed that a variety of substituents are tolerated on the dihydropyrimidine ring, indicating that the AII receptor is permissive in accepting this region of the nonpeptide antagonists. As reported for imidazole-based AII antagonists, the tetrazolyl dihydropyrimidine analogs were found to be more potent than the corresponding carboxylic acids. Our studies show that dihydropyrimidine analogs 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5-yl)[1, 1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester (Ki = 8.3 nM), 2-butyl-4-chloro-1,6-dihydro-6-methyl-1- [[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-5- pyrimidinecarboxylic acid (Ki = 1.0 nM), and 2-butyl-6-chloro-1,4-dihydro-4,4-dimethyl-1-[[2'-(1H-tetrazol-5-yl )[1,1'- biphenyl]-4-yl]methyl]-5-pyrimidinecarboxylic acid, ethyl ester (Ki = 1.1 nM), display affinities for the AII receptor which are comparable to or better than losartan (Ki = 9.0 nM). One of these derivatives, 2-butyl-4-chloro-1,6-dihydro-6-methyl-1-[[2'-(1H-tetrazol-5- yl)[1,1'-biphenyl]-4-yl]methyl]pyrimidine-5-carboxylic acid, ethyl ester, showed antihypertensive activity on oral administration to spontaneously hypertensive rats. These results demonstrate that the imidazole of losartan can be successfully replaced with a dihydropyrimidine ring.


A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.


Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet. This communication reports the kinetics of the Na+/Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1-10-microM range. The PM Ca(2+)-ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147-163) for [Ca2+]cyt < or = 1.5 microM with the fluorescent Ca2+ indicator quin2 (Kd = 115 nM). That study determined that the PM Ca2+ pump in the basal state has a Vmax = 0.098 mM/min, a Km = 80 nM and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (Kd = 500 nM), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the Kd and fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca(2+)-free medium (above citation) or (ii) by the newly developed "ionomycin short-circuit" method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a Km = 0.97 +/- 0.31 microM and Vmax = 1.0 +/- 0.6 mM/min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal Vmax of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt > or = 1.5 microM for 20-50 sec. Activation raises the pump Vmax to 1.6 +/- 0.6 mM/min and the Km to 0.55 +/- 0.24 microM. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mM in the 0.1 to 3 microM range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.


Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells. When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25 mM), taurine (19 mM) and glycine (9 mM). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na+ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mM for nine different amino acids in Na(+)-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K+ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.