Effect of aging on presynaptic alpha 2-adrenoceptor mechanisms in guinea pig ileum

1. Presynaptic alpha 2-adrenoceptor mechanisms in electrically stimulated longitudinal muscles of ilea isolated from 3, 10, 20 and 47 week-old guinea pigs were studied by analysis of the concentration-response curves of noradrenaline, a full agonist, and clonidine, a partial agonist, and the Scatchard plot of specific binding of [3H]-p-aminoclonidine to synaptosomal fractions from the longitudinal muscle of guinea pig ileum. 2. The pD2 value of noradrenaline and the maximum contraction induced by clonidine increased with age from 3 to 20 weeks and there after decreased to 47 weeks, while the pA2 value of yohimbine against noradrenaline did not alter with age. 3. The capacity of the maximum binding sites of [3H]-p-aminoclonidine increased with increasing age (3-20 weeks), while the dissociation constant (Kd) of [3H]-p-aminoclonidine did not change during the same period. 4. The changes in the presynaptic alpha 2-adrenoceptor mechanisms with age are considered to be due to the change in the total concentration of presynaptic of alpha 2-adrenoceptors.     

Responses of the isolated rectum of the rainbow lizard (Agama agama) to sympathomimetics

The relaxant effects of isoprenaline, noradrenaline, and adrenaline on the isolated rectum of the rainbow lizard (Agama agama) were studied. Responses were measured as a reduction of carbachol-induced contractions for each sympathomimetic agent. Isoprenaline, adrenaline, noradrenaline produced a dose-dependent relaxation of this preparation and the order of potency was as given. The pD2 value of 8.15 +/- 1.88 obtained for isoprenaline was significantly different (p less than 0.05) from those for adrenaline (5.80 +/- 0.90) and noradrenaline (5.25 +/- 1.18). H35/25, propranolol, and practolol competitively antagonized the relaxant effects of isoprenaline on the isolated lizard rectum. The pA2 values for these beta-adrenoceptor antagonists did not differ significantly (at p less than 0.05). alpha-Adrenoceptor antagonists, phentolamine and phenoxybenzamine, failed to alter the relaxant responses of these sympathometics to any appreciable extent. These results are interpreted to suggest that the relaxant effect produced by these sympathomimetics are mediated predominantly by beta-adrenoceptors that are not significantly differentiated into subtypes. alpha-Adrenoceptors in this preparation contribute minimally to the observed inhibitory response following sympathomimetic stimulation.     

Characteristics of beta-adrenoceptors in tracheal smooth muscle of guinea pig sensitized by egg albumin

The effect of pretreatment with egg albumin was examined on the beta-adrenoceptors in guinea pig isolated trachea. Befunolol and carteolol acted as partial agonists and their pA2 values were significantly larger than their corresponding pD2 values in tracheae from both untreated guinea pigs and those treated with egg albumin, suggesting that the beta-adrenoceptors contain two different affinity sites. The Scatchard plot of specific [3H]befunolol binding showed two affinity sites of the receptor (high and low affinity sites) in tracheae from both untreated animals and those treated with egg albumin. The pKD values of befunolol for both low and high affinity sites were in agreement with their respective pD2 and pA2 values. The intrinsic activities of befunolol and carteolol and the pD2 values of the test drugs were decreased by the treatment with egg albumin. The treatment with egg albumin also decreased the total amount of the two affinity sites of the receptor without any change in affinity. The present results support the partial blockade of beta-adrenoceptors in asthma proposed by Szentivanyi.     

Serotonin and histamine receptor-mediated inhibition of serotonin and noradrenaline release in rat brain cortex under nimodipine treatment

Rat brain cortex slices preincubated with [³H]serotonin or [³H]noradrenaline (25 100 nmol/l each) were superfused and the effects of serotonin and histamine on the electrically (0.3 or 3 Hz) evoked tritium overflow were studied.

In slices preincubated with [³H]serotonin the extent of inhibition of the electrically (3 Hz) evoked tritium overflow produced by histamine was increased when the concentration of [³H]serotonin used for incubation was decreased. The evoked overflow tended to be lower in slices from 2-year-old rats than in slices from 6-month-old animals whereas the inhibitory effect of histamine on the evoked overflow did not differ. Treatment of rats with nimodipine for at least 6 weeks did not significantly affect the evoked overflow in slices from 6-month and 2-year-old rats nor did it significantly alter the serotonin- and histamine-mediated inhibition of the evoked overflow in slices from young adult rats. The extent of histamine-mediated inhibition of the electrically evoked tritium overflow from slices (of young adult rats) preincubated with [³H]noradrenaline did not change when the concentration of [³H]noradrenaline used for incubation was decreased; the degree of inhibition markedly increased when the frequency of stimulation was lowered from 3 to 0.3 Hz. The inhibitory effect of histamine on the electrically (0.3 Hz) evoked overflow was mimicked by the H₃ receptor agonist R-(−)--methylhistamine and antagonized by the H₃ receptor antagonist thioperamide. The electrically evoked overflow and its inhibition by histamine were not affected by nimodipine, irrespective of whether the Ca²⁺ antagonist was administered in vivo (for at least 6 weeks) or added to the superfusion medium in vitro.

It is concluded that (1) the extent of the H₃ receptor-mediated effect in rat brain cortex slices can be markedly increased by lowering the concentration of the tracer in slices preincubated with [³H]serotonin and by lowering the stimulation frequency in slices preincubated with [³H]noradrenaline; (2) the H₃ receptor-mediated inhibition of serotonin release is not changed during aging and (3) nimodipine does not significantly influence serotonin release and noradrenaline release and their serotonin and/or histamine receptor-mediated modulation.     

Influence of the thromboxane-mimetic U-46619 and the prostacyclin metabolite 6-keto prostaglandin E1 on vasoconstriction induced by noradrenaline and 5-HT in rat mesenteric vasculature

The aim of this study was to investigate the effects of U-46619, a thromboxane A2-mimetic, and 6-keto prostaglandin E1 (6-keto PGE1) a biologically active metabolite of prostacyclin, on vasoconstrictor responses to noradrenaline and 5-hydroxytryptamine (5-HT). In vitro, U-46619 (3-100 nmol/l) amplified responses to both noradrenaline and 5-HT in a concentration-dependent manner. This effect was not caused by an increase in the affinity of the alpha-adrenoceptor for noradrenaline because U-46619 (100 nmol/l) did not alter the pA2 of phentolamine. In vivo, U-46619 (100 nmol/l) induced vasoconstriction and consequently significantly shifted the log-concentration-effect curves to noradrenaline and 5-HT upward in an additive manner. 6-Keto PGE1 (1 mumol/l) did not affect either perfusion pressure or vasoconstriction in response to noradrenaline in vivo. The study highlights some differences in responses between in vitro- and in vivo-perfused mesentery.     

Characterization of alpha1-adrenoceptor subtypes mediating noradrenaline-induced contraction of rat epididymal vas deferens in calcium-free medium.

The alpha1-adrenoceptor subtype mediating noradrenaline (NA)-induced contractions of rat epididymal vas deferens in Ca2+-free/EGTA (1 mM) medium was studied using competitive antagonists. The effects of chloroethylclonidine (CEC) was investigated in Ca2+-free and normal Krebs' medium and RT-PCR was used to identify alpha1-adrenoceptor specific mRNA in epididymal vas deferens. In Ca2+-free medium, NA evoked sustained contractions but was less potent (pD2, 5.9) than in normal Krebs' medium (pD2, 7.3). The contractions in Ca2+-free medium were inhibited by prazosin (pA2, 9.3), 5-methylurapidil (pA2, 8.4), spiperone (pA2, 7.6) and BMY 7378 (pK(B), 6.8) consistent with activation of alpha1A-subtype. Repeated pretreatment with CEC (100 microM) reduced the potency of NA and maximum contractions in normal and Ca2+-free media. CEC-sensitivity in normal Krebs' medium was enhanced by prior treatment with phenoxybenzamine. mRNA for alpha1a- and alpha1d- but not alpha1b-adrenoceptors were detected in epididymal vas deferens. These results suggest that NA contracts the tissue in Ca2+-free medium by the stimulation of alpha1A-adrenoceptors. Two factors affecting CEC-sensitivity of NA-induced contractions in this tissue are discussed.     

Decreased noradrenergic and serotonergic reactivity of vas deferens of newborn rats from mothers treated with the serotonin reuptake inhibitor fluoxetine during pregnancy and breast-feeding

Female Wistar rats were treated with the serotonin reuptake inhibitor fluoxetine (10 mg/kg/i.p/day), during pregnancy and breast-feeding, for the study of the corresponding newborn rats. At the end of the preweaning period, the 30-day old litters had their vas deferens removed for testing peripheral sympathetic reactivity, through the following experiments in vitro: (a) concentration-contraction curves for serotonin and for the adrenergic agonists noradrenaline, phenylephrine, clonidine and dopamine or for the indirect agonist tyramine (b) contractions induced by electric field stimulation, as an indicator of sympathetic neurotransmission (c) release of endogenous noradrenaline, measured by real-time determinations on HPLC (d) Ca(+2) time-contraction curves, to check for changes on Ca(+2) translocation. Our results showed that the affinity (pD(2)) for serotonin was strikingly decreased by about 1.5 log units. The pD(2) for adrenergic agonists was decreased by about 0.5 log units, except for dopamine and clonidine. The maximum effects and intrinsic activity were decreased only for dopamine. On the other hand, the response to Ca(+2) and the release of noradrenaline from nerve terminals were not modified. In additional experiments, the mother's body weights were measured, showing a decrease during gestation and a recovery during lactation while the offspring's weights were lower than controls. It is concluded that, besides the alterations on body weights, changes on noradrenergic and serotonergic mechanisms were observed and persisted in the newborn, at least one month after parturition.     

Effects of cooling on alpha-1-adrenoceptor mechanisms in rat aorta

1. The effects of cooling from 37 degrees C to 25 degrees C on alpha-1-adrenoceptor mechanisms in isolated rat aortic strips were studied. 2. The dissociation constant and the maximum binding for [3H]-prazosin, and the pA2-values (negative logarithm of dissociation constant) of clonidine and prazosin against noradrenaline were not influenced by cooling. 3. These results indicate that cooling did not influence the affinity of a competitive antagonist and receptor concentration. 4. Cooling increased the dissociation constant of noradrenaline but decreased its efficacy. 5. A receptor occupancy-response curve for noradrenaline was a rectangular hyperbola at 37 degrees C but linear at 25 degrees C, suggesting that a relationship between the contractile response and receptor occupancy was changed by cooling.     

Further characterisation of the dopamine-inhibitory receptor in Helix and evidence for a noradrenaline-preferring receptor

1. The cells in this study responded with a hyperpolarization to the following agents in this order of potency; dopamine greater than noradrenaline phenylephrine = octopamine. 2. 6,7 ADTN had a relative potency of 0.1 compared to dopamine. 5,6 ADTN did not inhibit the cells in this study. 3. The D1 receptor agonists SKF38393 and dihydroxynomifensine mimicked the effect of dopamine on these cells but were over 100 times less active, whereas the D2 selective agonists quinpirole and RU24213 were without effect. 4. Both the D1 antagonist SCH23390 and the D2 antagonist sulpiride antagonised the dopamine response with pA2 values of 6.1 and 6.7, respectively. 5. Five cells that responded to dopamine with a hyperpolarization were depolarized by noradrenaline. The order of potency of compounds at eliciting this depolarization, noradrenaline greater than phenylephrine greater than octopamine indicated that this response may be mediated by a noradrenaline-preferring receptor.     

Possible involvement of serotonin receptors in the facilitatory effect of a hallucinogenic phenethylamine on single facial motoneurons

2,5-Dimethoxy-4-methylamphetamine (DOM, "STP") is a potent hallucinogen, proposed to be a serotonin receptor agonist. Its effects have not previously been tested upon central neurons where serotonin is excitatory and serotonin antagonists are effective. Extracellular single unit recordings were obtained from facial motoneurons in anaesthetized rats, and drugs were applied from five-barrelled micropipettes by iontophoresis. Facial motoneurons were commonly silent. During subthreshold application of glutamate, firing could be induced by dopamine and DOM. As reported by others, serotonin and noradrenaline also excited facial motoneurons under these conditions. Methysergide antagonized responses to serotonin and DOM but not those to noradrenaline; methysergide could not usually discriminate between responses to serotonin and dopamine. Ketanserin reversibly antagonized (but could not discriminate between) responses to serotonin, dopamine, and noradrenaline. Chlorpromazine antagonized responses to dopamine at doses that did not alter serotonin-induced excitation, and responses to DOM were not reduced by doses of chlorpromazine, that had no local anaesthetic effect on action potentials elicited by DOM and serotonin. These results suggest that DOM is an agonist on at least one type of central serotonin receptor. This receptor may also be a ketanserin (5-HT2) binding site.     

Effect of different albumin media on the lipid-mobilizing action of sympathicotropic substances in vitro.

The authors studied the effect of adrenotropic substances on lipolysis in rat epididymal adipose tissue in albumin medium in vitro. On using albumins of different origin (human, bovine), the pD2 values for catecholamines differed by more than one order, in correlation to the type of albumin used. The isopropylnorsynephrine pD2 values did not differ. The addition of ascorbic acid (100 microng/ml) raised the catecholamine pD2 values and completely equalized the pD2 values found in both media. The pD2 values for the synephrine derivative did not alter. The propranolol pA2 values were not negatively affected by the addition of ascorbic acid. Ascorbic acid also produced a mild increase in the maximum lipid-mobilizing values obtained with any of the given substances in either medium. It was concluded in the discussion that catecholamines are oxidized at different rates in different albumin media and that this oxidation can be inhibited by adding ascorbic acid. Ascorbic acid likewise mildly stimulates the maximum lipid-mobilizing effect. The authors recommend the addition of ascorbic acid to albumin medium as a regular component for the study of adrenergic lipid mobilization.     

Responsiveness of the trimmed rat subcutaneous adipose tissue beta-receptors to catecholamines in ontogenesis

In order to estimate the possible changes of the functional state of beta-adrenergic receptors during ontogenesis, the lipid mobilizing effects of isoprenaline (ISO), adrenaline (ADR) and noradrenaline (NA) were studied in trimmed subcutaneous adipose tissue of rats at different stages of postnatal development (5, 14, 21 and 35 days after birth. The release of free acids (FFA) into the incubation medium was measured after addition of increasing concentrations of catecholamines and the dose-response curves were evaluated. In all the studied age groups, ISO produced the highest maximum effect and had the greatest potency. The calculated pD2 values indicate the same potency order ISO much greater than NA = ADR in all age groups and they also demonstrate that there exist no appreciable differences in the affinity single catecholamines at different stages of postnatal development. In comparison with all the other groups, the intrinsic activity of the studied adrenomimetics was relatively low in 14-day-old rats.     

Influence of estradiol and progesterone on the sensitivity of rat thoracic aorta to noradrenaline

The aim of this study was to investigate the effects of low and high doses of estradiol, and of progesterone on the response to noradrenaline in rat thoracic aorta. Two weeks after bilateral ovariectomy, female rats received a s.c. injection of vehicle (corn oil, 0.1 mL/day), estradiol (10 microg/kg/day or 4 mg/kg/day) and/or progesterone (20 mg/kg/day), for eight days. On the ninth day, the rats were sacrificed and aortic rings, with or without endothelium, were used to generate concentration-response curves to noradrenaline. Aortic rings with intact endothelium from the high-dose (4 mg/kg/day) estradiol group were supersensitive to noradrenaline compared to the vehicle or low-dose (10 microg/kg/day) estradiol groups (pD2 values = 7.86+/-0.09, 7.30+/-0.11 and 7.35+/-0.04, respectively). Endothelium-intact aortic rings from high-estradiol rats were supersensitive to noradrenaline when compared to vehicle-, progesterone- and progesterone + high-estradiol-treated rats (pD2 values = 7.77+/-0.12, 7.21+/-0.13, 6.93+/-0.04 and 7.22+/-0.18, respectively). There were no significant differences among the pD2 values for noradrenaline in aortic rings without endothelium. In conclusion, at high but not low doses, estradiol increased the sensitivity to noradrenaline and this was prevented by progesterone. Both of these effects were endothelium-dependent.     

Effect of morphine on the concentration of monoamines in the emotiogenic zones of the hypothalamus in adult and old rats

The content of monoamines in the emotiogenic hypothalamic zones has been shown to noticeably change with aging of rats. The level of noradrenaline and serotonin increased in the ventromedial nucleus of the hypothalamus, while the concentration of noradrenaline increased in the lateral hypothalamic zone. Single i.p. injections of 10 mg/kg morphine evoked qualitatively different shifts in the monoamine concentrations in the hypothalamic emotiogenic zones of the rats of different ages: the level of dopamine increased in adult animals, while the levels of noradrenaline and serotonin dropped in old rats. It is supposed that in old age the effect of morphine on dopaminergic structures in the emotiogenic hypothalamic zones becomes more moderate, whereas that on the noradrenergic and serotonergic structures is facilitated. The age-related specificities of the morphine effect on the monoaminergic regulation of the emotiogenic hypothalamic zones can determine considerable modifications of a psychotropic effect of the drug in old age.     

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.     

Restraint stress enhances morphine-induced analgesia in the rat without changing apparent affinity of receptor

Antagonism of morphine analgesia (tail-flick assay) by naloxone was assessed quantitatively by in vivo "apparent" pA2 determination in unstressed rats and in rats subjected to restraint stress. Restrained rats had a higher baseline tail-flick latency than did unstressed (unrestrained) animals, and were more sensitive to the analgesic effect of morphine, as reflected in lower morphine ED50s. There was no significant difference between apparent pA2 values of unstressed and restrained rats using pA2 regression line analysis. This suggests that while stress enhances the analgesic effect of morphine, it does not appreciably alter opiate receptor affinity for naloxone under the conditions of this study.     

Relationship between pD2 and pA2 of neuroleptics

The differences in the pD2/pA2 ratio of nine tested neuroleptics were found to vary between 0.92 and 1.11. This result confirms the hypothesis that pD2 (as determined from the inhibition of spontaneous locomotor activity) and pA2 (as determined from the shift of apomorphine dose response curve induced by a fixed dose of the neuroleptic) should not differ markedly and is discussed as differences in receptor specifities on the basis of the concept of multiple binding sites fo neuroleptics in the brain.     

Atrial supersensitivity to noradrenaline in stressed female rats

Stress can change the responses to catecholamines in many tissues. The aim of this study was to investigate the influence of the estrous cycle on the sensitivity of right atria to noradrenaline in female rats subjected to acute swimming stress. Female Wistar rats in proestrus, estrus, metestrus or diestrus were submitted to a 50 min-swimming session. Immediately after the exercise, the rats were killed and their right atria were mounted for isometric recording of the spontaneous beating rate. Concentration-effect curves to noradrenaline were obtained before and after the inhibition of neuronal uptake with phenoxybenzamine (10 microM) and of extraneuronal uptake with estradiol (5 microM). Acute swimming stress did not change the right atrial sensitivity to noradrenaline in rats in estrus, metestrus and diestrus. However, swimming stress produced supersensitivity to noradrenaline in proestrus (pD(2) control: 7.14 +/- 0.03 vs. pD(2) swimming: 7.55 +/- 0.04; p<0.05). This supersensitivity was still observed after uptake inhibition. When catecholamine uptake was inhibited, the concentration-effect curve to noradrenaline was shifted to the left 2.5-fold in the proestrus control group and 1.7-fold in the proestrus stress group (p<0.05). In conclusion, the estrous cycle influenced the acute stress-induced atrial supersensitivity to noradrenaline.     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.