Alterations of cholinergic receptors and the vesicular acetylcholine transporter after lateral fluid percussion injury in newborn piglets.

AIMS: Traumatic brain injury (TBI) is one of the leading causes of death and disability in children. Adult animal models of TBI showed cholinergic alterations. However, there is no comparable data on immature animals. Therefore, this study investigates cholinergic markers in a large animal model of juvenile TBI. METHODS: Twenty-seven female newborn piglets were subjected to lateral fluid percussion (FP) injury and compared with 12 untreated animals. After 6 h, animals were sacrificed and the brains removed. The hemispheres ipsilateral to FP-TBI from seven piglets and corresponding hemispheres from six control animals were used for autoradiography. Receptor density was determined with [(3)H]epibatidine (nicotinic acetylcholine receptors) or [(3)H]QNB (muscarinic acetylcholine receptors). The density of the vesicular acetylcholine transporter (vAChT) was assessed with (-)-[(3)H]vesamicol. Cerebral blood flow was measured by coloured microsphere method. RESULTS: Cerebral blood flow and brain oxygen delivery were transiently reduced early after FP-TBI (P < 0.05). TBI caused reductions of muscarinic acetylcholine receptor density (fmol/mg) in the basal forebrain (sham: 10797 +/- 1339, TBI: 8791 +/- 1031), while nicotinic acetylcholine receptor remained stable. Significant increases in vAChT density (fmol/mg) were observed in the basal forebrain (sham: 2347 +/- 171, TBI: 2884 +/- 544), putamen (sham: 2276 +/- 181, TBI: 2961 +/- 386), cortex (sham: 1928 +/- 262, TBI: 2377 +/- 294), thalamic areas (sham: 2133 +/- 272, TBI: 2659 +/- 413), hippocampus (sham: 2712 +/- 145, TBI: 3391 +/- 501) and hypothalamus (sham: 2659 +/- 139, TBI: 3084 +/- 304). CONCLUSIONS: Cholinergic markers are altered after mild-to-moderate TBI in the immature brain. Whereas the ACh receptors are stable in almost any brain region after TBI, vAChT expression increases after trauma at the employed severity of this specific trauma model.

Release of MCP-1 and IL-8 from lung epithelial cells exposed to volatile organic compounds.

Increased indoor air concentrations of volatile organic compounds (VOC) have been shown to contribute to the risk of respiratory and allergic diseases. The aim of this study was to investigate the inflammatory potential of single VOC and mixtures using an in vitro model. TNF-alpha stimulated human lung epithelial cells (A549) were exposed to VOC (1 ng/m3-100g/m3) via gas phase. After 20 h of exposure cytotoxicity and the release of the pro-inflammatory molecules monocyte chemoattractant protein-1 (MCP-1), Interleukin-6 (IL-6) and IL-8 was analysed. Exposure of A549 cells to chlorobenzene, styrene or m-xylene increased the MCP-1 production within the indoor relevant concentration range (1-25,000 microg/m3), higher concentrations increased the secretion of IL-8. Mixtures of aromatic compounds caused comparable effects to the single compounds on MCP-1 and IL-8 with a shift to lower concentration ranges. Neither the aliphatic compounds n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, and methylcyclopentane nor the mixture of these VOC showed any effects on MCP-1 and IL-8 production. Cytotoxic effects were not observed. These results show that aromatic, but no aliphatic compounds stimulate the release of pro-inflammatory mediators from lung epithelial cells. When aromatic compounds were mixed the sensitivity of lung cells to these compounds was increased.

Hypoxia and neuronal function under in vitro conditions.

Neurons in the mammalian CNS are highly sensitive to the availability of oxygen. Hypoxia can alter neuronal function and can lead to neuronal injury or death. The underlying changes in the membrane properties of single neurons have been studied in vitro in slice preparations obtained from various brain areas. Hypoxic changes of membrane potential and input resistance correspond to a decrease in ATP concentration and an increase in internal Ca2+ concentration. Functional modifications consisting of substantial membrane depolarization and failure of synaptic transmission can be observed within a few minutes following onset of hypoxia. The hypoxic depolarization accompanied by a hyperexcitability is a trigger signal for induction of neuronal cell death and is mediated mainly by activation of glutamate receptors. The mechanisms of the hypoxic hyperpolarization are more complex. Two types of potassium channels contribute to the hyperpolarization, the Ca(2+)- and the ATP-activated potassium channel. A number of neurotransmitters and neuromodulators is involved in the preservation of normal cell function during hypoxia. Therefore, hypoxia-induced cellular changes are unlikely to have a single, discrete pathway. The complexity of cellular changes implies that several strategies may be useful for neuroprotection and a successful intervention may be dependent upon drug action at more than one target site.

Adenosine A(1) and A(3) receptors mediate inhibition of synaptic transmission in rat cortical neurons.

Intracellular recordings were made in rat brain slice preparations containing pyramidal cells of the associative frontal cortex in order to characterize the action of selective adenosine A(1) and A(3) receptor ligands on synaptic neurotransmission. The selective A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) inhibited concentration-dependently the excitatory postsynaptic potentials (PSPs) which were evoked by focal electrical stimulation. The CPA-mediated inhibition was blocked by 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX), a highly A(1) receptor-selective antagonist. The A(3) receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) inhibited concentration-dependently the evoked PSPs while the A(1) receptors were blocked continuously by DPCPX. Under these conditions, the A(3) receptor antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino]-1,2,4-triazolo[1, 5-c]quinazoline (MRS 1220) did not influence the PSPs but inhibited completely the effect of IB-MECA. The inhibitory effect of IB-MECA was unaffected by DPCPX. CPA additionally inhibited the PSPs when applied after IB-MECA. Pharmacological dissociation of the N-methyl-D-aspartate (NMDA) and non-NMDA receptor components of the PSPs showed that CPA as well as IB-MECA reduced both. We conclude that adenosine A(1) and A(3) receptors are present on cortical pyramidal cells and involved in the inhibition of excitatory neurotransmission. Our results indicate no interplay between the two receptor subtypes. The separate inhibition may become particularly evident in situations where there are high levels of endogenously released adenosine, as seen in hypoxia.

Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat.

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of alpha,beta-methylene ATP (alpha,beta-meATP) caused reproducible depolarizations which were depressed by suramin (30 microM) and abolished by suramin (100 microM). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10, 30 microM) also concentration-dependently inhibited the alpha,beta-meATP-induced depolarization, although with a much slower time-course than suramin. Almost complete inhibition developed with 30 microM PPADS. Reactive blue 2 (30 microM) did not alter the effect of alpha,beta-meATP, while reactive blue 2 (100 microM) slightly depressed it. 2. Pressure-applied (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 microM) depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM) abolished the response to AMPA. Suramin (100 microM) potentiated the AMPA effect. 3. Pressure-applied noradrenaline hyperpolarized LC neurones. Suramin (100 microM) did not alter the effect of noradrenaline. 4. Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(-)-2-amino-5-phosphonopentanoic acid (AP-5; 50 microM), CNQX (50 microM) and picrotoxin (100 microM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 microM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP-5 (50 microM), CNQX (50 microM), picrotoxin (100 microM), strychnine (0.1 microM), tropisetron (0.5 microM) and hexamethonium (100 microM), a high concentration of suramin (300 microM) almost abolished the p.s.p. without changing the i.p.s.p. 5. In the presence of kynurenic acid (500 microM) and picrotoxin (100 microM), PPADS (30 microM) depressed the p.s.p. Moreover, the application of suramin (100 microM) to the PPADS (30 microM)-containing medium failed to cause any further inhibition. Neither PPADS (30 microM) nor suramin (100 microM) altered the i.p.s.p. 6. It was concluded that the cell somata of LC neurones are endowed with excitatory P2-purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co-transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

Effects of the central analgesic tramadol and its main metabolite, O-desmethyltramadol, on rat locus coeruleus neurones.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and, therefore, presumably additional mechanisms of analgesic action. Tramadol and its main metabolite O-desmethyltramadol were tested on rat central noradrenergic neurones of the nucleus locus coeruleus (LC), which are involved in the modulation of nociceptive afferent stimuli. 2. In pontine slices of the rat brain the spontaneous discharge of action potentials of LC cells was recorded extracellularly. (-)-Tramadol (0.1-100 microM), (+)-tramadol (0.1-100 microM), (-)-O-desmethyl-tramadol (0.1-100 microM) and (+)-O-desmethyltramadol (0.01-1 microM) inhibited the firing rate in a concentration-dependent manner. (+)-O-desmethyltramadol had the highest potency, while all other agonists were active at a similar range of concentrations. 3. (-)-Tramadol (10, 100 microM) was less inhibitory in brain slices of rats pretreated with reserpine (5 mg kg-1, 5 h before decapitation) than in controls. 4. The effect of (-)-tramadol (10 microM) was abolished in the presence of the alpha 2-adrenoceptor antagonist, rauwolscine (1 microM), whilst that of (+)-O-desmethyltramadol (0.3 microM) virtually disappeared in the presence of the opioid antagonist, naloxone (0.1 microM). (+)-Tramadol (30 microM) and (-)-O-desmethyl-tramadol (10 microM) became inactive only in the combined presence of naloxone (0.1 microM) and rauwolscine (1 microM). 5. In another series of experiments, the membrane potential of LC neurones was determined with intracellular microelectrodes. (-)-Tramadol (100 microM) inhibited the spontaneous firing and hyper-polarized the cells; this effect was abolished by rauwolscine (1 microM). (+)-O-desmethyltramadol (10 microM)had a similar but somewhat larger effect on the membrane potential than (-)-tramadol. The (+)-O-desmethyltramadol-(10 microM) induced hyperpolarization was abolished by naloxone (0.1 microM).6. The hyperpolarizing effect of noradrenaline (30 microM) was potentiated in the presence of (-)-tramadol(100 microM), but not in the presence of (+)-O-desmethyltramadol (10 microM). There was no potentiation of the noradrenaline (30 microM) effect, when the cells were hyperpolarized by current injection to an extent similar to that produced by (-)-tramadol (100 microM).7. Both noradrenaline (100 microM) and (- )-tramadol (100 microM) decreased the input resistance.8. The results confirm that the analgesic action of tramadol involves both opioid and non-opioid components. It appears that (-)-tramadol inhibits the uptake of noradrenaline and via a subsequent increase in the concentration of endogenous noradrenaline indirectly stimulates alpha2-adrenoceptors. (+)-0-desmethyltramadol seems to stimulate directly opioid micro-receptors. The effects of (+)-tramadol and(-)-O-desmethyltramadol consist of combined micro-opioid and alpha2-adrenergic components.

Effect of desamino-cholecystokinin-octapeptide (CCK-7) on the intraluminal pressure and myoelectrical activity of the gall-bladder, stomach, and small intestine in conscious dogs.

Desamino-cholecystokinin-octapeptide (CCK-7) increased the gall-bladder pressure and decreased the gastric pressure following i.v. infusion in conscious dogs. In small intestine and Heidenhain pouch CCK-7 exerted an initial excitatory effect followed by an inhibitory effect on intraluminal pressure and peristalsis. In all organs the effect of CCK-7 on pressure was correlated with a change of spike discharge. CCK-7 is more potent on gastrointestinal motor activity than a Boots preparation of pancreozymin. The results suggest that CCK-7 at doses within the physiological range could be involved in the regulation of gastrointestinal motility.

Activation of A(3) receptors by endogenous adenosine inhibits synaptic transmission during hypoxia in rat cortical neurons.

PURPOSE: The aim of our study was to characterize the influence of A(3) receptors on synaptic potentials (PSPs) in pyramidal cells from the rat cingulate cortex during hypoxia. METHODS: Intracellular recordings (n=49) were taken from slice preparations. PSPs were evoked by electrical stimulation. RESULTS: Hypoxia (95%N(2)-5%CO(2), 5 min) reduced the amplitude of the PSPs significantly. The effect was more pronounced in the presence of adenosine re-uptake inhibitor S-(p-nitrobenzyl)-6-thioguanosine (NBTG) and deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA); the effect was completely reversed by bovine adenosine deaminase. Hypoxic inhibition induced after A(1) receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in the presence of NBTG was completely reversed by the A(3) antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino]-1,2,4-triazolo[1,5-c]quinazoline (MRS 1220), indicating the involvement of A(3) receptors in hypoxic PSP inhibition. This was confirmed by A(3) agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) inhibiting PSPs. The effect of IB-MECA was blocked by the rat A(3) receptor-selective antagonist 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS 1523) and was not observed in the presence of G-protein inhibitor guanosine-5'-O-2-thiodiphosphate (GDP-beta-S). CONCLUSION: We conclude that a high level of endogenous adenosine, which occurs during hypoxia, activates A(3) receptors. Their activation contributes to PSP inhibition by adenosine during hypoxia.

Effect of an adenosine A(1) receptor agonist and a novel pyrimidoindole on membrane properties and neurotransmitter release in rat cortical and hippocampal neurons.

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.

Therapy of bronchial asthma with adenosine receptor agonists or antagonists.

Adenosine is an endogenous nucleoside that is released under pathological conditions and interacts with four G-protein-coupled receptor subtypes. These receptors are widely distributed throughout the body. They are involved in many central and peripheral processes, including immunological and inflammatory responses. In inflammatory and asthmatic conditions, the extracellular concentration of adenosine increases in the airway tissue. It enhances mast cell degranulation and bronchoconstriction, but may also inhibit eosinophil or lymphocyte function or modulate reactive oxygen species generation in neutrophils. Despite a large number of studies clearly indicating the effects of adenosine in vitro, many aspects of the mechanisms involved in the adenosine-mediated responses are still unclear, and our knowledge is limited in understanding the complex multifactorial interactions occurring in the whole body. The discovery of adenosine receptor compounds acting with increasing selectivity will bring new approaches to the use of adenosine receptor agonists and antagonists and may clarify some of the current uncertainties. On the basis of our present knowledge, the development of adenosine A(2A)- or (A3)-receptor agonists as antiinflammatory agents or A(2B)-receptor antagonists as inhibitors of mast cell degranulation for the treatment of asthma holds promise.

Tolerance to inhibition by ethanol of N-methyl-D-aspartate-induced depolarization in rat locus coeruleus neurons in vitro.

Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus. The pressure application of N-methyl-D-aspartate (NMDA) produced reproducible depolarizations of stable amplitude. Superfusion with ethanol (100 mM) for 15 min inhibited the depolarizing response to NMDA: the effect of ethanol was rapidly reversed on washout. When the superfusion time of ethanol (100 mM) was increased to 60 min, its inhibitory effect disappeared after 50 to 60 min. Moreover, after the subsequent washout of ethanol a withdrawal-like increase in the sensitivity to NMDA became evident. Hence, adaptive mechanisms of locus coeruleus neurons during the long-time contact with ethanol may be modelled in an in vitro system.

Neuroprotection by ATP-dependent potassium channels in rat neocortical brain slices during hypoxia.

Morphological changes induced by 30 min of hypoxia (incubation in medium saturated with 95% N2-5% CO2 instead of the normal 95% O2-5% CO2) were investigated in neurons (layers II/III of the parietal cortex) of rat neocortical brain slices. The cells were identified as intact, reversibly or irreversibly injured. As expected, hypoxia decreased the number of intact cells and increased the number of irreversibly injured cells. Pretreatment of slices with diazoxide (300 microM), an agonist of ATP-dependent potassium (KATP) channels completely prevented the morphological damage induced by hypoxia, whereas tolbutamide (300 microM), an antagonist of KATP channels, was ineffective when given alone. However, tolbutamide (300 microM) co-applied with diazoxide (300 microM), partly reversed the neuroprotective effect of this agonist during hypoxia. In conclusion, KATP channels appear to be present on neocortical neurons and their opening counteracts hypoxia-induced cell injury.

Neuropeptide Y potentiates via Y2-receptors the inhibitory effect of noradrenaline in rat locus coeruleus neurones.

Intracellular recordings were carried out in a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Pressure application of noradrenaline with various pulse durations inhibited the spontaneous frequency of action potentials and hyperpolarized the membrane. Neuropeptide Y (NPY), its C-terminal fragment NPY (16-36) and peptide YY (PYY), at a concentration of 0.1 mumol/l all, potentiated the effect of noradrenaline, while [Leu31, Pro34]NPY (0.1 mumol/l) was inactive. These results are compatible with the presence of Y2-type NPY-receptors at the cell somata of LC neurones.

Inhibition by ethanol of excitatory amino acid receptors in rat locus coeruleus neurons in vitro.

Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). In a first series of experiments, various parameters of spontaneous action potentials were evaluated. It turned out that ethanol (100 mM) does not alter the firing rate, the spike amplitude and the afterhyperpolarization following a spike. In subsequent experiments, the generation of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. Under these conditions, ethanol (100 mM) had no effect on the membrane potential or input resistance. Pressure-applied N-methyl-D-aspartate (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and alpha,beta-methylene ATP (alpha,beta-meATP) reproducibly depolarized LC neurons. While ethanol (100 mM) depressed the NMDA- and AMPA-induced depolarization to a similar extent, it did not interact with alpha,beta-meATP. Lower concentrations of ethanol (10 and 30 mM) had no effect on depolarizing responses to NMDA or AMPA. Noradrenaline applied by pressure pulses reproducibly hyperpolarized LC cells. These hyperpolarizations were unchanged by ethanol (100 mM). Biphasic synaptic potentials consisting of early depolarizing (PSP) and late hyperpolarizing (IPSP) components were evoked by electrical stimulation. Ethanol (100 mM) depressed the PSP and increased the IPSP. Glutamatergic PSPs recorded in the combined presence of picrotoxin (100 microM) and suramin (100 microM) were also inhibited by ethanol (100 mM). However, IPSPs recorded under these conditions were insensitive to ethanol (100 mM). In conclusion, ethanol may interfere with the AMPA (or NMDA) receptor-mediated fraction of the PSP and slightly facilitate the alpha2 adrenoceptor-mediated fraction of the IPSP.

Modulation of locus coeruleus neurons by extra- and intracellular adenosine 5'-triphosphate.

The cell membrane of rat locus coeruleus (LC) neurons is sensitive to both extra- and intracellular ATP. Extracellular ATP or its enzymatically stable analogues activate membrane receptors of the P2 type. These receptors inhibit a persistent potassium current and simultaneously activate a nonselective cationic conductance. The resulting depolarization increases the spontaneous firing rate. A decrease in the concentration of intracellular ATP during hypoxia or hypoglycemia opens ATP-sensitive K+ (KATP) channels of LC neurons. The resulting hyperpolarization depresses the discharge of action potentials and conserved energy. The hypoxia-induced hyperpolarization is additionally due to the release of adenosine from neighboring neurons or glial cells. A certain class of compounds, termed potassium channel openers, also decrease the firing, while sulphonylurea antidiabetics known to block KATP channels increase it. Sulphonylurea antidiabetics antagonize the excitability decrease induced both by potassium channel openers and metabolic damage.

Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K(ATP) channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K(ATP) channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K+,Na+-ATPase by limitation of energy supplying substrate.

Suc-Tyr(SE)-Met-Gly-Trp-Met-Asp-beta-phenethylamide(410): a competitive antagonist of cholecystokinin-induced contractions in smooth muscles in vitro.

Suc-Tyr(SE)-Met-Gly-Trp-Met-Asp-beta-phenethylamide(410) has been studied for its ability to antagonize contractile responses of guinea pig gall bladder, ileum and stomach muscle strips to desamino-cholecystokinin-octapeptide (CCK-7) and cholecystokinin octapeptide (CCK-8). Both CCK-7 and CCK-8 at concentrations of 10(-11)M to 10(-7)M produced dose-dependent tonic contractions in all muscle strips. Suc-Tyr(SE)-Met-Gly-Trp-Met-Asp-beta-phenethylamide (10(-8)M-10(-5)M) inhibited reversibly in a dose-dependent manner the contractile responses to CCK-7 and CCK-8. At the same concentrations the antagonist shifted to the right in parallel to the dose-response curves for CCK-7 and CCK-8 without decreasing their maximum response. Analysis of the data after Schild gave pA2 values (410 potency as antagonist) of CCK-7 in gall bladder, ileum and stomach of 8.36; 8.0 and 7.56, respectively, and pA2 values of CCK-8 of 7.64; 8.94 and 8.52, respectively. The slope of the Schild plots for both CCKs did not differ significantly from the unity, which suggests that 410 is a competitive antagonist. The antagonistic action of 410 is reversible and appeared to be specific since at concentrations of 5 X 10(-6), it had no effect on contractile responses of the gall bladder, ileum and gastric muscle strips to acetylcholine or histamine.

Responses of guinea-pig gastric, ileal and gall bladder smooth muscle to desamino-cholecystokinin-octapeptide (CCK 7).

Cholecystokinin 7 (CCK 7), a synthetic analogue of cholecystokinin/pancreozymin (CCK 33), increased in a dose-dependent manner the tone of the guinea-pig ileal, gastric and gall bladder smooth muscle preparations. In all these preparations CCK 7 was more potent than CCK 8 and CCK 33 and all three cholecystokinins were more potent than acetylcholine (ACH). Atropine and tetrodotoxin (TTX) did not influence the CCK 7 action in fundus and gall bladder muscle strips but reduced non-competitively its effect in ileal muscle strips. Neither GE 410 nor dbcGMP affected the ACH and histamine (His) response of the muscle strips but both antagonists shifted the dose-response curve of CCK 7 to the right, GE 410 (cholecystokinin antagonist) being a much more potent antagonist of CCK 7 as compared to dbcGMP. In all muscle strips a competitive action on the CCK 7 responses was found for GE 410. In gastric muscle strips a competitive influence on the CCK 7 responses was found for dbcGMP at low concentration (1 x 10(-5)M) and a non-competitive influence at high concentration (5 x 10(-4)M). The results suggest that the contractile effects of CCK 7 in the isolated ileal smooth muscle are realized by cholinergic and direct myogenic mechanisms, whereas in the isolated gall bladder and gastric smooth muscles, by a direct myogenic mechanism only.

[Differences in activity between substance P and substance P-heptapeptide as studied on the phenomenon of "gut dependence" in rats (author's transl)]. / Wirkungsunterschiede zwischen Substanz P und Substanz P-Heptapeptid, untersucht am Phänomen der "gut dependence" bei Ratten.

Naloxone causes a tonic contraction (= "gut dependence") of the ileum in morphine-dependent rats. This "gut dependence" can be antagonized by morphine (2.5 X 10(-7) g/ml). Substance P-undecapeptide (SP1-11) produces a tonic contraction of the ileum in morphine-independent rats and also in morphine-dependent rats. In the latter, this tonic contraction is comparable with the naloxone-induced contraction. When morphine-dependent rats with naloxone-induced "gut dependence" are given SP1-11, they show an additional increase in tonus followed by a rapid tonus inhibition. Substance P-heptapeptide (SP5-11) also causes a naloxone-like contraction in both the morphine-dependent and the morphine-independent rats. In contrast to SP1-11, SP5-11 produces neither a tonus superposition nor a tonus inhibition in morphine-dependent rats with naloxone-induced "gut dependence". The fact that SP5-11 does not, in contrast to SP1-11, exert these two effects (tonus superposition and subsequent tonus inhibition) is indicative of differences between the mechanisms of the two Substance P sequences.

Different action of substance P on gastric and ileal smooth muscle.

On the smooth muscle of the gastrointestinal system, substance P (SP) has both a stimulatory and an inhibitory effect. The effect depends on dose and route of application (in vivo or in vitro). In vivo (stomach and ileum of dog) at low dose, SP inhibits the amplitude and frequency of peristaltic contractions without influencing tone. In higher dose, SP stimulates the tone and inhibits peristaltic contractions. In vitro (circular preparations of fundus and corpus of guinea pig stomach and longitudinal preparations of guinea pig ileum), only the stimulatory effect of SP is found. At high SP concentrations, stimulation of tone is accompanied by a postexcitatory inhibition of peristaltic contractions. The results demonstrate that there are two types of inhibitory activity of SP on contractions of smooth muscles. One of these can be seen only during in vivo experiments at low concentrations. This primary inhibitory effect could be the result of the action of SP on the neuronal elements. The other kind of inhibitory effect on contraction is a secondary effect, and is only induced at high concentrations of SP and only in connection with a stimulatory effect on tone. The secondary inhibitory effect of SP may be connected with the direct effect of SP on smooth muscle cells.