Regional Comparison of Rat Colon Mechanics

The purpose of this study was to examine the force-velocity characteristics of colonie muscle and to determine whether these factors contribute to regional specialization of the colon observed in adult Fischer rats. Four measurements were obtained from circular colon muscle strips: maximum shortening velocity, a reflection of crossbridge cycling rate; extension of the parallel elastic component, a measure of passive muscle properties of relaxed muscle; the series elastic component, a measure of passive muscle properties of contracted muscle; and peak isometric force, which is equal to the product of the number of activated crossbridges and the strength of individual crossbridges. Muscle length (L) was expressed in terms of the length of optimal tension development (L_o). Peak isometric force and maximum shortening velocity were 848.9 ± 114.7 g/cm² and 0.082 ± 0.012 L_o/s for muscle strips from the proximal colon, and 948.0 ± 138.2 g/cm² and 0.083 ± 0.014 L_o/s for muscle strips from the distal colon. Shortening velocity, isometric force, and load extension properties of the parallel elastic component and the series elastic component are similar in proximal and distal rat colon. This suggests that regional specialization is not determined at the myofibril level but is most likely determined by extrinsic regulatory factors at the neural or receptor level.     

The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.     

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [³H]nimodipine and [³H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [³H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [³H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [³H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [³H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [³H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [³H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [³H]nimodipine and [³H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.     

Response of Smooth Muscle from Proximal and Distal Human Colon

Regional differences in colonic motility may be responsible for the orderly transit of intraluminal contents through the colon. The aims of this study were to compare the effect of stretch on active and passive stress development in colonic muscle from the proximal and distal colon and to compare the responses of these tissues to KC1 or bethanechol stimulation. Strips of taenia or circular smooth muscle were obtained from the disease-free segment of the colon removed for adenocarcinoma. Passive, active, and total isometric stress were measured on full-thickness strips of circular or longitudinal taenial muscle stimulated with bethanechol (10⁻⁴ M) as the muscles were stretched to 120% of the length of optimum tension ( L _o.) The tissues then were stimulated with increasing concentrations of KCI and bethanechol while being stretched at L _o. The active stress in the proximal circular muscle was greater at all levels of stretch than in distal circular or longitudinal muscle ( p <.001). The resting and passive stress were greater in distal circular and longitudinal taenial muscle than in proximal circular muscle ( p < .05). There was a dose-dependent increase in stress development to bethanechol and KCl in each type of muscle. Proximal circular muscle had the greatest response. The EDSO was shifted to the right in distal circular muscle (2.6 ± 0.1 × 10⁻⁵ M) compared to proximal circular muscle (1.1 ± 0.1 × 10⁻⁵ M) ( p < .001). These studies suggest that muscle stress differs in different locations of the colon and the role of active and passive stress development must be considered in models explaining in vivo colonic motility disturbances.     

HIV-1 Envelope Protein gp120 Potentiates NMDA-evoked Noradrenaline Release by a Direct Action at Rat Hippocampal and Cortical Noradrenergic Nerve Endings

Exposure of rat or human neocortical or hippocampal tissue to glutamate receptor agonists elicits a Ca²⁺-dependent, exocytotic-like release of previously accumulated [³H]noradrenaline through activation of both N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors colocalized on the noradrenergic axon terminals. Here we show that the NMDA (100 μM)-evoked release of [³H]noradrenaline from superfused thin layers of isolated rat hippocampal or cortical nerve endings was potentiated when the human immunodeficiency virus type 1 coat protein gp120 was added to the superfusion medium concomitantly with NMDA. The effect of gp120 (10 pM to 3 nM) on the 100 μM NMDA-evoked release of [³H]noradrenaline was concentration-dependent; the maximal effect (-140% potentiation) was reached at 100 pM of gp120. The protein was inactive on its own. The [³H]noradrenaline release evoked by NMDA (100 μM) + gp120 (100 μM) was prevented by classical NMDA receptor antagonists, as well as by 10 μM memantine. Neither the release evoked by NMDA nor that elicited by NMDA + gp120 was sensitive to the nitric oxide synthase inhibitor N ^G -nitro- l -arginine, suggesting no involvement of nitric oxide. The [³H]noradrenaline release elicited by 100 nM AMPA was unaffected by gp120. The protein potentiated the release evoked by 100 nM glutamate; the effect of 100 pM gp120 was quantitatively identical to that of 1 μM glycine, with no apparent additivity between gp120 and glycine. The antagonism by 1 μM 7-chloro-kynurenic acid of the NMDA-induced [³H]noradrenaline release was reversed by glycine or gp120. The data are compatible with gp120 acting directly as a powerful positive allosteric modulator at a neuronal NMDA receptor.     

Time Course of Dopamine-D2 and Serotonin-5-HT2 Receptor Occupancy Rates by Haloperidol and Clozapine In Vivo

Abstract: In vivo occupancy of dopamine-D₁, D₂ and serotonin-5-HT₂ receptors by haloperidol 10 mg/kg and clozapine 20 mg/kg were studied. Rats were injected intravenously with [³H]-YM-09151-2, [³H]-SCH23390, or [³H]-ketanserin 10 min after the administration of the tested drugs. Fifteen to 240 min after the ligand injection, the receptor occupancy rates of the drugs in the striatum and frontal cortex were calculated. Clozapine demonstrated the higher 5-HT₂ and lower D₂ occupancies in the respective regions. A dose-response analysis of D₂ and 5-HT₂, receptor occupancy by the drugs consolidated the higher 5-HT₂ binding affinity of clozapine in comparison with haloperidol. The present methodology may serve as an accurate tool to evaluate the peculiarity of various antipsychotics.     

Evidence For Two Neurochemical Divisions in the Human Nucleus Accumbens

The neurochemical anatomy of the human nucleus accumbens was studied by comparing the distributional patterns of [³H]DAMGE (μ opioid receptor), [³H]bremazocine (κ opioid receptor), [³H]SCH-23390 (D₁-like dopamine receptor), [³H]7-OH-DPAT (D₃ dopamine receptor) binding, preproenkephalin mRNA and acetylcholinesterase activity in sections of post mortem human striatum. Our results demonstrate the presence of at least two neurochemically distinct divisions within the human nucleus accumbens, which may be homologous to the 'shell'and'core'divisions of the nucleus as found in the rat.     

Transferrin receptors in the Parkinsonian midbrain

Several hypotheses have been put forward to explain the pathogenesis of Parkinson's disease (PD) and recently it has been suggested that alterations in iron homeostasis may be implicated. Because of the central role of the transferrin receptor in providing access of iron to cells, we have studied the distribution and density of transferrin receptors using [³H]–transferrin ([³H]–Tf) binding and tritium film autoradiography in the normal and PD midbrain. High levels of [³H]–Tf binding were found in the dorsal raphé, oculomotor nucleus and periaqueduc tal grey whilst lower levels of [³H]–Tf binding were found in the tegmentum, red nucleus and substantia nigra. Significant reductions in binding were found in the substantia nigra, red nucleus and oculomotor nucleus in PD, the reductions in [³H]–Tf binding being similar to the loss of nigral neurons in PD. The data suggest that the increased iron content of surviving nigral neurons may reflect a compensatory metabolic response rather than abnormal transferrin receptor expression.     

Inhibition of Brain Oestrogen Receptors by RU 58668

The purpose of this study was to determine if injections of the 11ß-substituted steroidal antioestrogen, RU 58668, would block two measures of oestrogen receptor action in ovariectomized adult female rats. Using an in vitro nuclear exchange assay, it was found that RU 58668 reduced cell nuclear []> ³H]-oestradiol binding in brain tissue 24  h after it was injected. However, pituitary cell nuclear [³H]-oestradiol binding was significantly reduced just 2  h after the antioestrogen was injected. Our results demonstrate that RU 58668 can reach the brain following a subcutaneous injection, but that it needs more time to reach the brain than it does to reach the pituitary. Since the levels of hypothalamic and pituitary progestin receptors are known to be regulated by oestradiol, cytosolic [³H]-R5020 binding was used as an in vitro assay of endogenous oestrogen receptor action. RU 58668 blocked induction by oestradiol of cytosolic [³H]-R5020 binding in both the brain and pituitary 48  h after it was injected. In the absence of oestradiol, RU 58668 did not stimulate cell nuclear [³H]-oestradiol binding or cytosolic [³H]-R5020 binding in either brain or pituitary. In conclusion, injections of RU 58668 blocked two measures of oestrogen receptor action in the brain and pituitary without showing oestrogenic activity itself.     

Transferrin receptors in the normal human hippocampus and in Alzheimer's disease

Several lines of evidence suggest that aluminium may play a role in the pathogenesis of Alzheimer's disease (AD). The iron transport protein transferrin is the major transport protein for aluminium, and aluminium gains access to cells by means of a specific cell surface transferrin receptor. We have assessed the distribution of transferrin receptors in the normal and AD hippocampal formation using [³H]–transferrin ([³H]–Tf) binding and tritium film autoradiography, in order to assess the role of the transferrin receptor in AD. In normal brain, ³H]–Tf binding was highest in the pyramidal cell layers with CA2> dentate gyrus granule cell layer ≥Cal >CA3 ≥ CA4>subiculum>parahippocampal gyrus. In AD, significant reductions in [³H]–Tf binding were found in CA1, CA2 and CA4 pyramidal cell layers. The reduced [³H]–Tf binding in AD may, however, be due to poor pre–mortem agonal states which correlated with reduced [³H]–Tf binding. The discrepancy between the distribution of transferrin receptors in the hippocampus and those areas which are prone to the formation of senile plaques and neurofibrillary tangles suggests that if transferrin–mediated uptake of aluminium in AD/SDAT is significant in the pathogenesis of this disorder, it is not the only determinant of Alzheimer–type neuropathology.     

β-Carbolines: Endogenous Ligands for the Benzodiazepine Receptor

Abstract: The interaction of benzodiazepines (BDZ) and β -carbolines with metal cations was investigated. Δmong the numerous transition metal cations, only four, Co²⁺, Ni², Cu²⁺ and Zn²⁺, specifically affected the receptor binding of [³H]diazepam and [³H] β -carboline-3-carboxylic acid ethyl ester ( β -CCE). The effects of these cations on [³H]diazepam binding were exactly opposite to those on [³H] β -CCE binding. An intraperitoneal injection of β -carboline-3-carboxylic acid methyl ester ( β -CCM) produced spike discharges and β-CCM induced spike discharges were prevented by an intramuscular injection of BDZ. These findings suggest that β -carbolines could be related to the proposed endogenous ligand and that BDZ might be antagonistic rather than agonistic of the naturally occurring ligand.     

A Comparative Study of α2- and β-Adrenoceptor Distribution in Pigeon and Chick Brain

The pharmacological properties and anatomical distribution of α₂, β₁ and β₂-adrenoceptors in pigeon and chick brains were studied by both homogenate binding and tissue section autoradiography. [³H]Bromoxidine (α₂-adrenoceptor-), [³H]CGP 12177 (β-adrenoceptor) and [¹²⁵1]cyanopindolol (β-adrenoceptor) were used as radioligands. In both species, [³H]bromoxidine binding to avian brain tissue showed a pharmacological profile similar to that previously reported for α₂-adrenoceptors in mammals. Regarding the anatomical distribution, the areas with the highest densities of α₂-adrenoceptors in the pigeon brain included the hyperstriatum, nuclei septalis, tectum opticum and some brainstem nuclei. Most β-adrenoceptors found in tissue membranes and sections from chick and pigeon brain were of the β₂ subtype, in contrast to what has been reported in the mammalian brain, where the β₁ subtype is predominant. A striking difference was found between the two species regarding the densities of these receptors: while pigeon brain was extremely rich in [¹²⁵1]cyanopindolol binding throughout the brain (mainly cerebellum) in the pigeon, the levels of labelling in the chick brain were much lower; the exception was the cerebellum, which displayed a higher density than other parts of the brain in both species. Overall, our results support the proposed anatomical equivalences between a number of structures in the avian and mammalian encephalon.     

Reserpine Affects Differentially the Density of the Vesicular Monoamine Transporter and Dihydrotetrabenazine Binding Sites

We have studied the effect of a single injection of reserpine (5 mg/kg, s.c.) on the synaptic vesicle monoamine transporter (VMAT) density in the rat striatum, using two labelling procedures: radioimmunolabelling with an antibody against VMAT, and binding of the specific ligand [³H]dihydrotetrabenazine ([³H]TBZOH). In the rostral and medial striatum, the distribution of VMAT immunoreactivity displayed the highest density in the lateral subregions. In the caudal part of the striatum, VMAT immunoreactivity showed increasing density from dorsal to ventral subregions. The VMAT immunoreactivity was not altered 2 and 30 days after the reserpine injection, whereas [³H]TBZOH binding site density, measured on adjacent slices, showed a dramatic decrease at day 2 and a moderate recovery at day 30, suggesting that despite a persistent blockade of [³H]TBZOH binding sites, VMAT protein density was unchanged.     

Effect of Forced-Running Stress on β-Adrenergic Receptors in Rat Brain Regions and Liver

Abstract: Rats were exposed to forced-running stress for 1 day, 3 days or a long term (approximately 2 weeks), and P-adrenergic receptor binding was then assayed using [³H]dihydroalprenoloI (DHA) in six brain regions and the liver. In the pons + med.obl., hypothalamus and midbrain, a reduction in Fadrenergic receptor density was first evident on day 1. In contrast, a decrease in Fadrenergic receptor density in the cerebral cortex and hippocampus was first evident on day 3. Decreased [³H]DHA binding in the pons + med.obl., cerebral cortex and hippocampus subsequently plateaued for the duration of the forced-running stress. In the midbrain and hypothalamus, however, decreased [³H]DHA binding subsequently returned to control levels despite the exposure to the forced-running stress. In the cerebellum and the liver, [³H]DHA binding did not change significantly throughout the stress. These results indicate that the forced-running stress induces both the time- and region-specific changes in Fadrenergic receptors. Moreover, the rats showed either a behavioral depression or a spontaneous recovery of running activity during the 2 weeks following the end of the long-term stress. Thus, we also examined the relationship of P-adrenergic receptors to these behavioral differences. [³H]DHA binding for the behavioral depression group was lower in the hippocampus and higher in the liver than for the spontaneous recovery group.     

Plasticity of &mgr; and delta Opioid Receptors in the Superficial Dorsal Horn of the Adult Rat Spinal Cord Following Dorsal Rhizotomies: A Quantitative Autoradiographic Study

The aim was to study the regulation of μ and δ opioid binding sites in the superficial layers (laminae I–II) of the dorsal horn of the adult rat spinal cord 1, 2, 4 and 12 weeks after unilateral dorsal rhizotomies of various extents. Using quantitative autoradiography and highly selective tritiated opioid ligands, we have shown that the decrease in [³H]Tyr*- d -Ala-Gly-NMe-Phe-Gly-ol ([³H]DAMGO) (μ sites) and [³H]Tyr*- d -Thr-Gly-Phe-Leu-Thr ([³H]DTLET) (δ sites) binding in the side ipsilateral to the lesion as compared to the intact side is related to the number of dorsal roots cut. In the segment central to the lesion, 1 week after the lesion, ipsilateral/contralateral side binding ratios for [³H]DAMGO were 0.70, 0.49, 0.36 and 0.25 when 1, 3, 5 and 7 roots respectively were sectioned. For [³H]DTLET, the ratios were 0.71, 0.54, 0.42 and 0.39. The time-related analysis of binding ratios showed that, in partially deafferented spinal segments after long-term deafferentation (12 weeks postlesion) there were greater numbers of μ and δ binding sites than in cases of short-term deafferentation (1–2 weeks). By contrast, in spinal segments considered as completely deafferented, there was no difference in the remaining μ and δ binding sites at 12 weeks as compared to 1 week postlesion. Consequently, it is deduced that the partial recovery of μ and δ binding observed after long-term partial deafferentation could be associated with neuronal plasticity (probably collateral sprouting) of fine diameter primary afferent fibres arising from intact dorsal roots.     

Changes in Cholinergic Markers Following Kainic Acid Lesion of the Ventral Globus Pallidus in Rat

Abstract: Choline acetyltransferase (CAT) and glutamate decarboxylase (GAD) activities and [³H] quinuclidinyl benzilate ([³H]QNB) binding were determined in the rat frontal cortex following damage to the basal forebrain cholinergic system. Pre- and postsynaptic changes in the cholinergic system with the passage of time werealso studied. After a unilateral injection of kainic acid into the right ventral globus pallidus, the GAD levels remained unaffected, but the CAT levels decreased to 63.4% after 7 days. After 12 weeks, the CAT levels had returned to 87% of the control value. The Umaxof [³H]QNB binding for the muscarinic receptor was higher in the ipsilateral cortex up to 4 weeks. On the other hand, the K_D value at 12 weeks was higher without a change in the Bmax of the [³H]QNB binding.
These findings might indicate an ongoing compensatory receptor mechanism of denervation supersensitivity as a response to early changes in presynaptic cholinergic activity and the production of postsynaptic effect with presynaptic cholinergic damage over a long period of time.     

Effects of the Anticonvulsant Losigamone and Its Isomers on the GABAA Receptor System

Summary: We conducted in vitro studies to clarify the possible involvement of GABA, receptor-mediated processes in the anticonvulsant effects of losigamone and its optical isomers AO-242 (+losigamone) and AO-294 (-losigamone). In binding experiments with cortical and cerebellar membrane preparations of rat brain, ≤100 μM losigamone did not affect the specific binding of [³H]GABA, [³H]flunitrazepam, or [³⁵S]t-butyl-bicyclo-phosphorothionate (TBPS) to their receptors. Losigamone, however, in concentrations of 10-8-10-5 M, stimulated ³⁶Cl influx into spinal cord neurons in the absence of exogenous GABA. This effect was inhibited by the GABA antagonists bicuculline (BIC) and picrotoxin (PIC). Losigamone 10^-5 M potentiated the effect of a suboptimal concentration of exogenous GABA M on 3³⁶Cl influx. Both isomers of losigamone likewise stimulated 36Cl influx into spinal cord neurons, and these effects were similarly antagonized by BIC and PIC. Losigamone and its optical isomers AO-294 and AO-242 antagonized potassium-induced hyperexcitability in rat hippocampal slices concentration dependently. There were no clear differences in the potencies of losigamone, AO-242, or AO-294. However, AO-294 and AO-242 differed significantly in their ability to suppress TBPS- induced hyperexcitability of hippocampal slices. Such observations demonstrate that although losigamone does not bind to GABA, benzodiazepine (BZD) or PIC binding sites of the neuronal chloride channel, it is capable of stimulating ³⁶Cl influx in the spinal cord neurons by a GABA-sensitive mechanism and at a side distant from the GABA channel.     

Distribution of Nicotinic Receptors in the Human Hippocampus and Thalamus

Neuronal nicotinic acetylcholine receptors consist of different subunits, α and β, with different subtype arrangement corresponding to distinct pharmacological and functional properties. The expression of α3, α7 and β2 mRNA in the human brain was studied by in situ hybridization and compared to [³H]nicotine, [³H]cytisine and [¹²⁵l]α-bungarotoxin binding in contiguous sections. The β2 probe showed a strong hybridization signal in the granular layer of the dentate gyrus and in the CA2/CA3 region of the hippocampus and in the insular cortex, and a signal of lower intensity in the subicular complex and entorhinal cortex. The α3 probe showed strong hybridization in the dorsomedial, lateral posterior, ventroposteromedial and reticular nuclei of the thalamus, and a weak signal in the hippocampal region and in the entorhinal, insular and cingular cortex. The amount of α7 mRNA was high at the level of the dentate granular layer and the CA2/CA3 region of the hippocampus, in the caudate nucleus and in the pulvinar and ventroposterolateral nuclei of the thalamus. [³H]Nicotine and [³H]cytisine binding appeared to be identical in anatomical distribution and relative intensity. It was high in the thalamic nuclei, the putamen and in the hippocampal formation in the subicular complex and the stratum lacunosum moleculare. The level of [¹²⁵l]α-bungarotoxin binding was particularly high in the hippocampus and in the pyramidal cells of the CA1 region, but was relatively low in the subicular complex. Our data indicate that in the human brain nicotinic receptor subtypes have discrete distributions, which are in part different from those of other species.     

Visual Activation in α-Chloralose-anaesthetized Cats Does Not Cause Lactate Accumulation in the Visual Cortex as Detected by [1HINMR Difference Spectroscopy

The hypothesis that neuronal activation results in lactate accumulation due to mismatch between glucose and oxygen consumption was tested in the cat model of visual activation by monitoring cerebral metabolism with localized ¹H nuclear magnetic resonance spectroscopy (MRS). Adult cats were anaesthetized with α-chloralose, paralysed and mechanically ventilated. Visual evoked potentials measured over the occipital cortex showed maximal amplitude at 2 Hz stimulation, but the latencies of the early cortical potentials, N1 and P1, were independent of stimulation frequency. High signal-to-noise ratio, short echo time volume-selected [¹H]MRS was used to monitor cerebral lactate with a temporal resolution of 70 s. Difference proton spectroscopy unambiguously showed no lactate peak in the visual cortex during visual activation at stimulation frequencies ranging from 1 to 16 Hz. Absence of change in lactate concentration during visual stimulation was confirmed by averaging all the spectra acquired during activation and subtracting them from reference spectra collected in darkness, a procedure that had a calculated lactate detection limit of 0.17 mM. We also reduced the O₂ in the inspired air to 13%, which decreased pO₂ from 94.5 ± 8.9 to 47.0 ± 6.8 mmHg, during visual stimulation at 2 or 4 Hz. At this low pO₂ level, visual stimulation did not cause lactate accumulation in the visual cortex, however. The present data show that neuronal activation to this degree in the cat brain is not associated with aerobic lactate production to an extent that can be detected with ¹H MRS.