Presynaptic inhibition of synaptic transmission in the rat hippocampus by activation of muscarinic receptors: involvement of presynaptic calcium influx

1. Modulation of presynaptic voltage-dependent calcium channels (VDCCs) by muscarinic receptors at the CA3–CA1 synapse of rat hippocampal slices was investigated by using the calcium indicator fura-2. Stimulation-evoked presynaptic calcium transients ([Ca_pre]_t) and field excitatory postsynaptic potentials (fe.p.s.ps) were simultaneously recorded. The relationship between presynaptic calcium influx and synaptic transmission was studied.
2. Activation of muscarinic receptors inhibited [Ca_pre]_t, thereby reducing synaptic transmission. Carbachol (CCh, 10 μM) inhibited [Ca_pre]_t by 35% and reduced fe.p.s.p. by 85%. The inhibition was completely antagonized by 1 μM atropine. An approximate 4^th power relationship was found between presynaptic calcium influx and postsynaptic responses.
3. Application of the N-type VDCC-blocking peptide toxin ω-conotoxin GVIA (ω-CTx GVIA, 1 μM) inhibited [Ca_pre]_t and fe.p.s.ps by 21% and 49%, respectively, while the P/Q-type VDCC blocker ω-agatoxin IVA (ω-Aga IVA, 1 μM) reduced [Ca_pre]_t and fe.p.s.ps by 35% and 85%, respectively.
4. Muscarinic receptor activation differentially inhibited distinct presynaptic VDCCs. ω-CTx GVIA-sensitive calcium channels were inhibited by muscarinic receptors, while ω-Aga IVA-sensitive channels were not. The percentage inhibition of ω-CTx GVIA-sensitive [Ca_pre]_t was about 63%.
5. Muscarinic receptors inhibited presynaptic VDCCs in a way similar to adenosine (Ad) receptors. The percentage inhibition of ω-CTx GVIA-sensitive [Ca_pre]_t by Ad (100 μM) was about 59%. There was no significant inhibition of ω-Aga IVA-sensitive channels by Ad. The inhibitions of [Ca_pre]_t by CCh and Ad were mutually occlusive.
6. These results indicate that inhibition of synaptic transmission by muscarinic receptors is mainly the consequence of a reduction of the [Ca_pre]_t due to inhibition of presynaptic VDCCs.

     

Renal effects of intrathecally injected tachykinins in the conscious saline-loaded rat: receptor and mechanism of action

1. The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β-Ala⁸]NKA (4–10) and [MePhe⁷]neurokinin B (NKB) at T₁₃ thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK₁ (RP 67580), NK₂ (SR 48968) and NK₃ (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function.
2. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3 μmol min⁻¹, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0 μl min⁻¹, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6 μmol min⁻¹, P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8 mmHg at 1 min and +6.3±2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes.
3. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2 μl min⁻¹, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2 μmol min⁻¹, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4 μmol min⁻¹, P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK₂ ([β-Ala⁸]NKA(4-10)) and NK₃ ([MePhe⁷]NKB) receptors were devoid of renal effects.
4. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK₁ receptors and the sympathetic renal nerve.

     

Spatial heterogeneity of the effects of calcitonin gene-related peptide (CGRP) on the microvasculature of ligaments in the rabbit knee joint

1. Experiments were performed in anaesthetized rabbits to examine the effects of calcitonin gene-related peptide (CGRP) and the CGRP antagonist CGRP_8–37 on blood flow to the medial collateral ligament of the knee joint.
2. Topical application of CGRP (10⁻¹³ to 10⁻⁹  mol) to the exposed external surface of eight knee joints resulted in dose-dependent dilatation of vessels in both the ligament and the joint capsule. The magnitude of this response varied significantly in different regions of the medial collateral ligament, with the 10⁻⁹  mol dose of CGRP giving the maximum response (101.5±25.3% increase) at the femoral insertion site of the medial collateral ligament and lowest (23.1±8.8%) at the tibial insertion site.
3. Topical application of CGRP_8–37 (0.1, 1 and 10  nmol) produced dose-dependent constriction of vessels in the ligament and the joint capsule in five knees, with a trend towards the greatest effect occurring at the femoral insertion site (45.8±8.1% reduction in blood flow). With the 10  nmol dose, the vasoconstrictor response at the femoral insertion site differed significantly (P<0.05) from the responses obtained at the tibial insertion and joint capsule sites.
4. Topical application of CGRP_8–37 (0.1, 1 and 10  nmol) to four chronically denervated knees produced substantially smaller vasoconstrictor responses at all sites. At the femoral insertion site, where 10  nmol CGRP_8–37 normally produces a 45.8±8.1% reduction in blood flow (n=8), ten days following denervation this response was reduced to 6.5±6.1%, this difference being significant (P=0.01).
5. Adrenaline was applied topically to augment blood vessel tone, in order to establish how effectively co-administration of CGRP would offset this increase in tone. Adrenaline (10⁻¹⁰  mol) produced vasoconstriction at all sites (n=6). In the capsule this vasoconstriction was virtually abolished when CGRP (10⁻⁹  mol) was co-administered with adrenaline but in the ligament vasodilatation occurred at all sites. This vasodilatation was significantly greater at the femoral insertion site compared to the tibial insertion and mid ligament sites (P<0.05 for both) and the capsule (P<0.01).
6. Topical application of substance P (10⁻¹⁰ or 10⁻⁹  mol) failed to elicit dilatation of ligament blood vessels.
7. These results suggest that endogenous CGRP may play an important role in regulating blood flow to different structures in and around the knee joint.

     

The inflammatory lesion of T cell line transferred experimental autoimmune encephalomyelitis of the Lewis rat: distinct nature of parenchymal and perivascular infiltrates

We have investigated the T cell receptor (TCR) repertoire in the inflammatory infiltrates of T line-transferred experimental autoimmune encephalomyelitis (EAE) of the Lewis rats. Using a panel of TCR V-specific monoclonal antibodies (mAbs) and immunocytochemistry, we studied the nature of the T cells entering the central nervous system (CNS) after transfer of either myelin basic protein (MBP)-reactive, or MBP-reactive but non-encephalitogenic T cell lines. All the MBP-specific T cell lines predominantely used the V8.2 TCR chain. T cell lines specific for the tuberculin purified protein derivative (PPD), using TCR V genes different from V8.2, served as controls. We first studied the time course of T cells entering the CNS. In all recipient rats, small, but significant numbers of -TCR-expressing infiltrate cells appeared in the CNS within the first 24 h after T cell transfer. In animals injected with either type of MBP-reactive T cells, the early infiltrate cells were preferentially located within the parenchyma of the spinal cord, while in PPD T lineinjected rats, the lymphocytes were mostly found in the meninges. TCR V gene usage was examined on the peak of clinical disease. Six days after T cell transfer, the TCR repertoire used by infiltrating lymphocytes in general seemed to be highly diverse. None of the V isotypes examined (i.e. V8.2, V8.5 or V10) was used by a major population of the -TCR-positive T cells. A more detailed, quantitative analysis of individual infiltrate compartments revealed, however, a preferential accumulation of V8.2-positive T cells within the parenchyma. In contrast, perivascular infiltrating cells used V genes randomly. Our results confirm first that activated T lymphocytes enter the brain rapidly irrespective of their antigen specificity. Second, the data show that most of the perivascular infiltrate T cells in the acute EAE lesion are host-derived, recruited presumably from the recirculating T cell pool, while the encephalitogenic, V8.2-positive T cells preferentially persist within the parenchyma.Abbreviations EAE experimental autoimmune encephalomyelitis - MBP myelin basic protein - TCL T cell line Supported by the Brazilian Research Council (CNPq)     

流行性出血热患者早期免疫应答的研究

应用反向被动血凝集抑制试验和血凝集抑制试验检测流行性出血热患者早期血清（７病日内）中病毒核蛋白、膜蛋白抗体。１０３例患者血清,仅核蛋白抗体出现者３３例,病情转径及无变化者３１例（９３．９４％）．而转重者仅２例（６．０６％）,且其血清抗体主要为ＩｇＧ型。仅膜蛋白抗体出现的患者９例,病情转重者８例（８８．８９％）,其抗体主要为ＩｇＭ．核蛋白、膜蛋白抗体均阳性的６１例患者,病情转归介于上述二者之间．提示：核蛋白抗体先出现的患者,病情多转轻,而膜蛋白抗体先出现的患者,病情普遍转重,似与病毒的＂一次性打击＂有关。在仅核蛋白抗体出现的患者中,有２例病情转重,但其抗体球蛋白类型为ＩｇＧ,故尚不能排除本病有＂二次感染＂的可能,这一现象在推行本病预防接种时值得重视,另对进一步探讨本病的发病机理及预测患者的病情转归亦具参考价值。     

Presynaptic opioid receptors on dopaminergic nerves in the rabbit caudate nucleus: coupling to pertussis toxin-sensitive G-proteins and interaction with D2 autoreceptors?

Slices of the rabbit caudate nucleus, preincubated with [³H]dopamine and subjected to electrical field stimulation, were used (1) to investigate the involvement of G-proteins in the signal transduction of presynaptic D₂ (auto)receptors and -opioid receptors on dopaminergic axon terminals in this tissue and (2) to study a possible mutual interaction of these two presynaptic receptors. Pretreatment of the slices with either pertussis toxin (8 g/ml; 18 h), or N-ethylmaleimide (30 M, 30 min) significantly reduced the inhibitory effects of both the D₂ agonist quinpirole and the -opioid receptor agonist U-50488H on the [³H]overflow evoked by 36 pulses (2 ms, 24 mA, 0.3 Hz), suggesting the coupling of both receptors to G-proteins.Experiments designed to study possible interactions of these two presynaptic receptors were carried out under stimulation conditions (only 1 pulse), which strongly diminish interference of endogenous transmitters released in the tissue with modulatory effects of exogenous drugs. For instance, due to the presence of endogenous dopamine, quinpirole was much less potent during 36-pulse-than during 1-pulse field stimulation, whereas the D₂ antagonist domperidone was almost without effect in the latter case. Using the 1-pulse stimulation paradigm, the concentration/response curve of quinpirole was unaffected in the presence of the halfmaximal inhibitory concentration of U-50,488 H (0.1 M). On the other hand, also quinpirole at its halfmaximal inhibitory concentration (0.1 M), hardly affected the concentration/response curve of U-50,488 H: only high concentrations of U-50,488 H (above 1 M) seemed to be slightly less effective in the presence than in the absence of the D₂ agonist. U-50,488 H, at these high concentrations, was also less potent under 36-pulse than under 1-pulse stimulation conditions. From these findings, we conclude that there is only a limited interaction between presynaptic D₂ autoreceptors and -opioid receptors on dopaminergic axon terminals in the rabbit caudate nucleus, despite they are both coupled to PTX/NEM-sensitive G-proteins. Correspondence to: R. Jackisch at the above address     

Receptor binding assays in analysing the bioavailability and pharmacodynamic bioequivalence of active drug moieties

The bioavailability and pharmacodynamic bioequivalence of a conventional and an experimental sustained-release formulation of 100 mg metoprolol tartrate were studied in a randomised cross-over study in seven healthy volunteers by assessing over 24 h the plasma kinetics of R,S-metoprolol, its ₁-adrenoceptor binding component, and by determining the extent to which the active drug moiety in plasma occupied rabbit lung ₁-and rat reticulocyte ₂-adrenoceptors.The formulations differed markedly in their kinetic characteristics: the peak plasma concentration (C_max) of R,S-metoprolol after administration of the conventional formulation was 140 ng·ml^–1, (n=7) and it was approximately one-third of that after the sustained-release formulation, 49 ng·ml^–1, (n=6); the AUC_{0–24 h}-values for the formulations were 700 and 310 ng·h·ml^–1, respectively. The C_max for the ₁-adrenoceptor binding component of metoprolol was 180 ng·ml^–1 (n=7) after administration of the conventional, and 74 ng·ml^–1 after administration of the sustained-release formulation. The corresponding AUC_{0–24 h}-values for the receptor binding component were 920 and 470 ng·h·ml^–1 (n=7).Thus, the kinetic differences between R,S-metoprolol and the ₁-receptor binding component were considerable and they were affected by the type of formulation. In general, after administration of the sustained-release formulation, the percentage ₁- and ₂-adrenoceptor occupancy of metoprolol in plasma was 5–15% less than after administration of the conventional formulation. At 0.5–1.5 h after drug intake the average ₁-adrenoceptor occupancy of the conventional formulation varied between 80–90% and that of the sustained release formulation between 20–76%. At these times the differences in receptor occupancy were significant; at 0.5–2 h after drug intake the average ₂-adrenoceptor occupancy of the conventional formulation varied from 20–30%, and that of the sustained-release formulation was 2–17%. At other times the difference in receptor occupancy between the formulations was not significant.The results demonstrate that plasma concentration-kinetics were more discriminating than -adrenoceptor-binding in analysing bioequivalence. It was possible to determine the bioavailability of the active ingredient of metoprolol and to study pharmacodynamic bioequivalence by using receptor binding assays.     

Effects of acute selective 5-HT1, 5-HT2, 5-HT3 receptor andα 2 adrenoceptor blockade on naloxone-induced antinociception

Several studies have demonstrated a paradoxical form of antinociception induced by the repeated administration of opioid antagonists accompanied by exposure to a painful stimulus. The underlying mechanism of this naloxone-induced antinociception (NIA) is still unknown, but the results of several studies suggest that it is a non-opioid response. This study was designed to investigate serotonergic and noradrenergic involvement in NIA. Rats were treated daily with systemic injections of 5 mg/kg naloxone, followed by a 45-s hot plate test of nociception (temperature=51.5 ± 0.5°C). After rats reached plateau levels of NIA, they received a test trial in which they were treated with various doses of different selective 5-HT or ₂ adrenoceptor antagonists in addition to naloxone before the hot plate test. Rats treated with 0.16, 0.32 and 0.63 mg/kg pirenperone or 2.5 mg/kg ritanserin showed significant reductions in paw lick latency with respect to rats treated with vehicle. In addition, high doses of yohimbine (7.5–10 mg/kg) also effectively reversed NIA. In contrast, NIA was not affected by acute blockade of 5-HT₁ or 5-HT₃ receptors by methiothepin or MDL 72222, respectively, or by the ₂ adrenoceptor blocker idazoxan. None of the 5-HT or ₂ adrenoceptor antagonists had any effect on the paw lick latencies of saline-treated rats. A possible role of 5-HT₂ receptors in the antinociception induced by opioid receptor blockade is discussed.     

Neuronal mechanisms of the attentional dysfunctions in senile dementia and schizophrenia: two sides of the same coin?

Deficits in early stages of information processing, specifically the inability to disattend irrelevant stimuli and to selectively allocate processing resources (i.e., hyperattention), have been associated with the development of psychotic symptoms. Opposite deficits, i.e., the failure to attend and select stimuli, and to divide attention (i.e., hypoattention), represent a major variable in the development of dementia. The hypothesis that hyperattention and hypoattention are mediated via cortical cholinergic hyperactivity and hypoactivity, respectively, is discussed. Several lines of evidence support the role of cholinergic hyperactivity in the development of psychotic symptoms, including the therapeutic effects of anticholinergic drugs in schizophrenic patients, the psychotic effects of chronic exposure to irreversible cholinesterase inhibitors, and the worsening of psychotic symptoms as a result of the treatment with cholinomimetic compounds. The potent impairments of attentional abilities as a result of the administration of muscarinic antagonists in intact subjects, and the attentional effects of cholinomimetic compounds in demented patients are two examples of the evidence that supports the role of cholinergic hypofunction in the cognitive impairments of dementia. A neuronal model of dopamine-GABAergic modulation of cortical acetylcholine is proposed on the basis of evidence indicating that nucleus accumbens dopamine, via a GABAergic pathway to the substantia innominata of the basal forebrain, modulates cortical acetylcholine release. The available evidence confirms several predictions derived from this model, including the dopaminergic regulation of cortical acetylcholine (ACh) release, the bidirectional modulation of this release by benzodiazepine receptor (BZR) agonists and inverse agonists, and the antipsychotic effects of BZR agonists. Bidirectional deviations in the activity of cortical cholinergic inputs are hypothesized to represent a major neuronal substrate of the attentional dysfunctions associated with, or even underlying, the development of psychotic symptoms and dementia. The walk of a stranger on the street could be a sign to me which I must interpret. Every face in the windows of a passing streetcar would be engraved on my mind, all of them concentrating on me and trying to pass me some sort of message. McDonald N (1960) Living with schizophrenia. Can Med Assoc J 82:218–227 Today my mother did not recognize me. Dette U (1991) Ein langer Abschied. Der Verlauf einer Alzheimer-Krankheit. (A long farewell. A case of Alzheimer's disease). Fischer Taschenbuch, Frankfurt [in German]     

Effects of chronic treatment with valproate on serotonin-1A receptor binding and function

Valproate is effective in treating bipolar disorder characterized by rapid cycling or acute mania, although the mechanism of action is unclear. In contrast to other treatments for depression, 21 days of treatment in rats with valproate (100, 200 or 400 mg/kg) did not significantly alter the hypothermia induced by 8-hydroxy-2-(di-n-propyl)aminotetralin (8-OH-DPAT), an agonist at serotonin-1A receptors. Treatment with valproate also had no effect on radioligand binding to serotonin-1A, serotonin-2 or -adrenergic receptors. Based on these animal studies in frontal cortex and hippocampus, the therapeutic benefit of valproate in mood disorders does not appear to involve adaptive changes in serotonin-1A, serotonin-2 or -adrenergic receptor number.