Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices

The presynaptic neuromodulation of stimulation-evoked release of [3H]-acetylcholine by endogenous adenosine, via A1-adenosine receptors, was studied in superfused hippocampal slices taken from 4-, 12- and 24-month-old rats. 8-Cyclopentyl-1,3-dimethylxanthine (0.25 microM), a selective A1-receptor antagonist, increased significantly the electrical field stimulation-induced release of [3H]-acetylcholine in slices prepared from 4- and 12-month-old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1-adenosine receptors. In contrast, 8-cyclopentyl-1,3-dimethylxanthine had no effect in 24-month-old rats. 2-Chloroadenosine (10 microM), an adenosine receptor agonist decreased the release of [3H]-acetylcholine in slices taken from 4- and 12-month-old rats, and no significant change was observed in slices taken from 24-month-old rats. In order to show whether the number/or affinity of the A1-receptors was affected in aged rats, [3H]-8-cyclopentyl-1,3-dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2-year-old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age-related increase in the adenosine level was found. In summary, our results indicate that during aging the level of adenosine in the extracellular fluid is increased in the hippocampus. There is a downregulation and reduced responsiveness of presynaptic adenosine A1-receptors, and it seems likely that these changes are due to the enhanced adenosine level in the extracellular space.     

Positive chronotropic and negative inotropic effects induced by potassium chloride in the isolated canine atrium

The effects of potassium chloride on inotropic and chronotropic activity were investigated in five isolated canine atrium preparations which were suspended in a bath and perfused with arterial blood from the carotid artery of the heparinized support dog. Potassium chloride administered into the cannulated sinus node artery in a dose range of 100 mug-1 mg produced a dose-related negative inotropic and a positive chronotropic effect. These effects were not influenced by treatment with either atropine or propranolol. From these results, it is concluded that potassium had a direct negative effect on atrial contractility and a direct positive effect on atrial rate.     

(+-)-N6-endonorbornan-2-yl-9-methyladenine (N-0861) and its enantiomers: selective antagonists of A1-adenosine receptors in guinea pig isolated atria

Adenosine and its metabolically stable analog 5'-N-ethylcarbox-amidoadenosine (NECA) induce negative inotropic, chronotropic and dromotrpic actions in the heart through activation of A1-adenosine receptors and relaxation of vascular smooth muscle through activation of A2-adenosine receptors. In vitro studies were carried out in order to determine the potency of the antagonist (+-)-N6-endonorbornan-2-yl-9-methyladenine (N-0861) and its two component enantiomers, WRC-0006(+) and WRC-0007(-), at the A1 receptors in the guinea pig atria and the A2 receptors in the guinea pig aorta. N-0861 competitively antagonized the negative inotropic responses induced by NECA in the eletrically paced left atrium (pKB = 6.24) and the negative chronotropic responses induced by NECA in the spontaneously beating right atrium (pKB = 6.29). WRC-0007 was 4-fold more potent (pKB = 6.51) than WRC-0006 (pKB = 5.86) at antagonizing the A1-adenosine receptors in the guinea pig left atrium. N-0861, WRC-0007 and WRC-0006 at high concentrations (> 3 x 10(-5) M) produced direct relaxations of the guinea pig aorta that masked to a small extent the A2 receptor antagonism by these compounds. The affinities of the antagonists for the A2 receptor in the aorta were calculated using the method of pharmacological resultant analysis. N-0861 was 47-fold less potent at the A2 receptor (pKB = 4.57) than it was at the A1 receptor. WRC-0006 was 2-fold more potent (pKB = 4.81) than WRC-0007 (pKB = 4.52) at the A2-adenosine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative regulation of MAP kinase by diacylglycerol-dependent mechanisms via G protein-coupled receptors in rat basophilic RBL-2H3 (ml) cells

Carbachol and 5'-(N-ethylcarboxamido)-adenosine (NECA), stimulants of G protein-coupled receptors, induce MAP kinase activation in the muscarinic ml receptor-transfected mast cell line, RBL-2H3 (ml) cells. The phospholipase C inhibitor neomycin and the phosphatidate phosphohydrolase inhibitor propranolol augmented MAP kinase activation induced by carbachol and NECA without affecting the antigen-induced MAP kinase activation. Furthermore, the duration of MAP kinase activation induced by carbachol or NECA was also prolonged by neomycin and propranolol. The specific protein kinase C inhibitor Ro 31-8425 enhanced the carbachol- or NECA-induced MAP kinase activation. These findings suggest that the MAP kinase activation mediated by the G protein-coupled receptors is negatively regulated by diacylglycerol and activated protein kinase C(s).     

Direct antagonism of ethanol''s effects on GABA(A) receptors by increased atmospheric pressure

We investigated hepatic blood flow, O2 exchange and metabolism in porcine endotoxic shock (Control, n = 8; Endotoxin, n = 10) with administration of hydroxyethylstarch to maintain arterial pressure (MAP)>60 mmHg. Before and 12, 18 and 24 h after starting continuous i.v. endotoxin we measured portal venous and hepatic arterial blood flow, intracapillary haemoglobin O2 saturation (Hb-O2%) of the liver surface and arterial, portal and hepatic venous lactate, pyruvate, glycerol and alanine concentrations. Glucose production rate was derived from the plasma isotope enrichment during infusion of [6,6-2H2]-glucose. Despite a sustained 50% increase in cardiac output endotoxin caused a progressive, significant fall in MAP. Liver blood flow significantly increased, but endotoxin affected neither hepatic O2 delivery and uptake nor mean intracapillary Hb-O2% and Hb-O2% frequency distributions. Endotoxin nearly doubled endogenous glucose production rate while hepatic lactate, alanine and glycerol uptake rates progressively decreased significantly. The lactate uptake rate even became negative (P<0.05 vs Control). Endotoxin caused portal and hepatic venous pH to fall significantly concomitant with significantly increased arterial, portal and hepatic venous lactate/pyruvate ratios. During endotoxic shock increased cardiac output achieved by colloid infusion maintained elevated liver blood flow and thereby macro- and microcirculatory O2 supply. Glucose production rate nearly doubled with complete dissociation of hepatic uptake of glucogenic precursors and glucose release. Despite well-preserved capillary oxygenation increased lactate/pyruvate ratios reflecting impaired cytosolic redox state suggested deranged liver energy balance, possibly due to the O2 requirements of gluconeogenesis.     

Agonist and antagonist actions of (-)pindolol at recombinant, human serotonin1A (5-HT1A) receptors

Prenatal diagnosis and clinical follow up of a patient with mosaicism for anomalies of chromosome 18 are reported. The fetus appeared on ultrasound to have multiple anomalies, including clubbed feet, abnormal hand positioning, edema of the scalp, cleft palate, and polyhydramnios. The karyotype on amniocytes was 47,XY,+i(18p). Postnatally, the peripheral blood karyotype was 46,XY,+i(18q), whereas the skin fibroblast karyotype was 47,XY,+i(18p). The infant had many features consistent with those previously described in cases of tetrasomy 18p and some that were consistent with trisomy 18q.     

Spillover-mediated transmission at inhibitory synapses promoted by high affinity alpha6 subunit GABA(A) receptors and glomerular geometry

Divergence and convergence of synaptic connections make a crucial contribution to the information processing capacity of the brain. Until recently, it was thought that transmitter released at a synapse affected only a specific postsynaptic cell. We show here that spillover of inhibitory transmitter at the Golgi to granule cell synapse produces significant cross-talk to non-postsynaptic cells, which is promoted both by the anatomical specialization of this glomerular synapse and by the presence of the high affinity alpha6 subunit-containing GABA(A) receptor in granule cells. Cross-talk is manifested as a novel slow rising and decaying small amplitude inhibitory postsynaptic current (IPSC) that can also contribute a long-lasting component to more typical IPSCs, which is prolonged by inhibition of the neuronal GABA transporter GAT-1. Because of the long duration of IPSCs generated by spillover, the total charge carried is three times that of IPSCs generated by directly connected terminals. GABA spillover within the mossy fiber glomerulus may play an important role in regulating the number of granule cells active in the cerebellar cortex, a regulation that is suggested by theoretical models to optimize cerebellar information processing.     

Disposition of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) in rats

The disposition of diadenosine 5'5"'-P1,P4-tetraphosphate (Ap4A), an endogenous dinucleotide, was investigated in rats. The degradation of Ap4A in rat plasma was very rapid and could be explained by a Michaelis-Menten equation: Km and Vmax values were 1.69 micrograms/ml and 4.32 micrograms/min/ml, respectively. Ap4A was degraded in rat plasma to ATP and AMP, but not to 2 ADP molecules, and these nucleotides were further degraded through adenosine. The degradations kinetics were examined. After intravenous bolus injection, Ap4A in plasma declined rapidly and the rate of elimination was dose-dependent: the biological half-life was about 3s at the dose of 1 mg/kg and was longer at 3 mg/kg. When Ap4A was administered by intravenous infusion (0.5 or 1.0 mg/kg/min), the plasma level rapidly reached a steady-state, which then rapidly declined after stopping the infusion.     

Endothelin ET(B) receptors counteract venoconstrictor effects of endothelin-1 in anesthetized rats

The inhibitory effects of BQ 788 (3 mg/kg, i.v., ET(B)-receptor antagonist) on endothelin-1 (ET-1)- or IRL 1620 (ET(B)-receptor agonist)-induced changes in mean arterial pressure (MAP), mean circulatory filling pressure (MCFP, driving force of venous return), arterial resistance (RA), venous resistance (RV) and cardiac output (CO) were characterized in 6 groups of pentobarbital-anesthetized rats. ET-1 or IRL 1620 (0.5, 1 and 2 nmol/kg, i.v.) dose-dependently increased MAP, RA, RV and MCFP and decreased CO. Maximum changes in RA, RV and CO elicited by ET-1 were greater than those by IRL 1620. Equimolar doses of ET-1 and IRL 1620 also caused similar initial transient decreases in MAP. BQ 788 alone slightly elevated MCFP, but did not alter other variables. The ET(B)-blocker abolished all changes elicited by IRL 1620, but only partially inhibited its responses on MCFP, showing the presence of BQ 788-insensitive receptors. BQ 788 also abolished ET-1's depressor response, partially inhibited its effect on MCFP, and markedly augmented its effects on RA, RV and CO. Thus, ET(B)-receptors counteract the sustained constrictor effects of ET-1 on arterial and venous resistance vessels Our results indicate a substantial arterial and venous dilator role for ET(B) receptors.     

Activation of BK(Ca) channel via endothelin ET(A) receptors in porcine coronary artery smooth muscle cells

It has been demonstrated previously that endothelin-1 stimulates the Ca2+-activated K+ (BK(Ca)) channel activity in porcine coronary artery smooth muscle cells. The purpose of the present study was to delineate the endothelin receptor subtype involved in this action. In receptor binding studies, [125I]endothelin-1 was shown to bind to the homogenate of porcine primary coronary artery smooth muscle cells in a single class of binding sites with K(D) and Bmax values of 73 pM and 99 fmol/mg protein, respectively. Furthermore, endothelin-1 and endothelin-3 displaced the binding of [125I]endothelin-1 to these cells with respective IC50 values of 70 and 17000 pM, a 240-fold difference in potency. The effects of endothelin-3 on the activity of the BK(Ca) channel in porcine coronary artery smooth muscle cells were examined using the cell-attached patch-clamp technique. Similar to endothelin-1, endothelin-3 also exhibited a bell-shaped concentration-response curve. A maximal increase of 95% in channel open-state probability (Po) was induced by 100 nM endothelin-3 as compared with the 320% increase in Po by 1 nM endothelin-1. Thus, endothelin-1 was about 100-fold more potent and 3.4-fold more efficacious than endothelin-3 in this action. Both the receptor binding and the electrophysiological results suggest that the effects of endothelins on the BK(Ca) channel are mediated through the endothelin ET(A) receptor subtype.     

8-(3-Chlorostyryl)caffeine (CSC) is a selective A2-adenosine antagonist in vitro and in vivo

An adenosine antagonist, 8-(3-chlorostyryl)caffeine (CSC), was shown previously to be 520-fold selective for A2a-adenosine receptors in radioligand binding assays in the rat brain. In reversing agonist effects on adenylate cyclase, CSC was 22-fold selective for A2a receptors in rat phenochromocytoma cells (Kb 60 nM) vs. A1 receptors in rat adipocytes (Kb 1.3 microM). Administered i.p. in NIH mice at a dose of 1 mg/kg, CSC shifted the curve for locomotor depression elicited by the A2a-selective agonist APEC to the right (ED50 value for APEC shifted from 20 micrograms/kg i.p. to 190 micrograms/kg). CSC had no effect on locomotor depression elicited by an ED50 dose of the A1-selective agonist CHA. CSC alone at a dose of 5 mg/kg stimulated locomotor activity by 22% over control values. Coadministration of CSC and the A1-selective antagonist CPX, both at non-stimulatory doses, increased activity by 37% (P < 0.001) over CSC alone, suggesting a behavioral synergism of A1- and A2-antagonist effects in the CNS.     

A potent and selective inhibition of parainfluenza 1 (Sendai) virus by new 6-oxiranyl-, 6-methyloxiranyluracils, and 4(3H)-pyrimidinone derivatives

Several new 6-oxiranyl-, 6-methyloxiranyluracils, and pyrimidinone derivatives, synthesized by the lithiation-alkylation sequence of 1,3,6-trimethyluracil, 1,3-dimethyl-6-chloromethyluracil, and 2-alkoxy-6-methyl-4(3H)-pyrimidinones, showed a potent and selective antiviral activity against the parainfluenza 1(Sendai) virus replication.     

Rapid oscillations in plasma glucagon-like peptide-1 (GLP-1) in humans: cholinergic control of GLP-1 secretion via muscarinic receptors

The mechanisms involved in the rapid glucagon-like peptide-1 (GLP-1) release following glucose ingestion are poorly defined. Besides a direct intestinal stimulation of L cells, humoral and neuronal mechanisms have been discussed. We investigated the temporal pattern of GLP-1 release in five healthy men (aged 27.8 +/- 3.6 yr, body mass index, 23.4 +/- 1.2 kg/m2) after an overnight fast for 60 min under basal conditions and for 60 min after an oral glucose load (OGL; 100 g) in both the presence and absence of atropine (80 ng/kg min, iv). Blood was sampled every 2 min, and data were evaluated for the temporal pattern of GLP-1 secretion by several computer-assisted programs (deconvolution, Pulsar analysis, and Fourier transformation). With all methods a pulsatile pattern of plasma GLP-1 levels with a frequency of five to seven per h was detected; this remained unchanged in the different metabolic states and during atropine treatment. Glucose and GLP-1 plasma levels showed a parallel increase after OGL (OGL without atropine = control: 8.4 +/- 2.9 and 7.9 +/- 3.0 min, respectively). Atropine infusion delayed this increase significantly (16.8 +/- 8.07 and 17.4 +/- 6.61 min, respectively; P < 0.02). In contrast to plasma glucose concentrations (82.7 +/- 0.3% of control; P < 0.05), atropine infusion reduced the integrated GLP-1 pulse amplitude to 56.0 +/- 11.3% of the control levels (P < 0.05). In conclusion, GLP-1 is secreted in a pulsatile manner with a frequency comparable to that of pancreatic hormones. Mean GLP-1 plasma concentrations increase after OGL due to augmented GLP-1 pulse amplitudes but not frequency. The differential effect of atropine on glucose and GLP-1 plasma levels suggest a direct cholinergic muscarinic control of L cells.     

Endothelin-1 induces vasoconstriction and nitric oxide release via endothelin ET(B) receptors in isolated perfused rat liver

Endothelin-1 (0.1, 1 and 10 nM) induced a significant increase in portal pressure and nitric oxide (NO) release in the isolated rat liver. The endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala(11,15)]endothelin-1-(8-21)) (0.1, 1 and 10 nM) also elicited a marked increase in portal pressure and NO release. The potency of endothelin-1 was higher than that of IRL 1620. The endothelin ET(A) receptor antagonist, BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu)) (1 and 10 microM), had no effect on the endothelin-1-induced change in portal pressure and NO current. In contrast, the endothelin ET(B) receptor antagonist, BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-++ +methoxycarbonyltryptophanyl-D-norleucine) (1 and 10 nM), attenuated the endothelin-1-induced change in portal pressure and NO current. Administration of N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, completely abolished the endothelin-1- or IRL 1620-induced NO release. L-NMMA enhanced the increase in portal pressure and decrease in O2 consumption caused by endothelin-1. These results indicated that endothelin ET(B) receptors mediate both vasoconstriction and NO release and that NO plays a significant role in stabilizing microcirculation in isolated perfused rat liver.     

Comparative pharmacology of recombinant rat AT1A, AT1B and human AT1 receptors expressed by transfected COS-M6 cells

1. Currently available antagonists and agonists cannot distinguish between angiotensin AT1 receptor subtypes. 2. We synthesized a series of compounds selected on the basis of having the most diverse structural features with respect to losartan (DuP753), the prototype non-peptide AT1 receptor antagonist. Using a radioligand-receptor binding assay and membranes prepared from COS-M6 cells transfected with individual AT1 receptor subtypes, we determined whether any of these compounds could distinguish between the receptor subtypes. 3. The diversity of the structural features of this series of compounds was reflected by the wide range of affinities (pIC50 values) displayed towards competing with [125I]-Sar1Ile8 angiotensin II for binding to the AT1 receptors. 4. Direct comparisons of the pIC50 values of individual compounds for rat AT1A, AT1B and human AT1 receptors revealed only minor differences. 5. It is concluded that compounds based structurally on losartan are unlikely to distinguish between these receptors.     

Level of interleukin-6 (IL-6), soluble interleukin-6 receptors (sIL-6R) and tumor necrosis factor alpha (TNF-alpha) in untreated and progressing multiple myeloma]

The effect of original analogues of the 6-9 C-terminal AVP fragment, D-MRP and D-MPRG, on the active avoidance behaviour of rats was studied using intranasal administration in a wide range of doses. The dose in 0.1 mcg/kg was the most efficient for D-MPR and 0.01 mcg/kg for D-MPRG, which for the tripeptide was ten times, and for the tetrapeptide hundred times lower than the corresponding dose of AVP used in the similar experiments. The tri- and tetrapeptide were shown to facilitate the active avoidance behaviour affecting both the formation of the reaction and consolidation of memory trace. The peptides mostly affect the perception processes, i.e., the detection of a specific stimulus from the environment, the estimation and memorizing of its biological significance.