Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions.

Based on an unexpected high maximum response to piperidine-4-sulphonic acid (P4S) at human alpha1alpha6beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABA(A) receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine-4-sulphonic acid (P4S) as partial agonists, respectively, at alpha1beta2gamma2, alpha6beta2gamma2 and alpha1alpha6beta2gamma2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole-cell patch-clamp recordings were used to investigate the pharmacological profile of the partial GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP), P4S, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), and 3-(4-piperidyl)isoxazol-5-ol (iso-4-PIOL), and the competitive GABA(A) receptor antagonists Bicuculline Methbromide (BMB) and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly alpha1, alpha2, alpha3, and alpha5 containing receptors and alpha1, alpha6 and alpha1alpha6 containing receptors, respectively. 4-PIOL and iso-4-PIOL, which at GABA(A) receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4-PIOL was slightly more potent at cortical neurones than at granule neurones and iso-4-PIOL was more potent in inhibiting isoguvacine-evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4-PIOL corresponded to that of a partial agonist, whereas that of iso-4-PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.

Effect of acute and long-term treatment with 17-beta-estradiol on the vasomotor responses in the rat aorta.

1. This study sought to evaluate whether the effects of acute and long-term treatment with 17-beta-estradiol on the vasomotor responses in rat aortic rings are mediated through the same mechanism. 2. Ovariectomized rats were treated daily with either 17-beta-estradiol-3-benzoate (100 microg kg(-1)) or vehicle for 1 week. 3. The effect of long-term 17-beta-estradiol treatment on the responses to cumulative doses of phenylephrine, 5-HT, calcium, potassium and 17-beta-estradiol was determined in aortic rings. In the same rings, the effect of acute exposure to 17-beta-estradiol (5 and 10 microM) on the dose response curves for phenylephrine, 5-HT, calcium, potassium and acetylcholine were estimated. The measurements were made in rings with and without intact endothelium. The tone-related basal release of nitric oxide (NO) was measured in rings with intact endothelium. 4. Long-term 17-beta-estradiol treatment reduced the maximum developed contraction to all contracting agents studied. This effect was abolished in endothelium denuded vessels. Acute 17-beta-estradiol treatment also reduced maximal contraction. This effect, however, was independent of the endothelium. 5. Long-term 17-beta-estradiol treatment significantly increased the ability of the rings to dilate in response to acetylcholine whereas acute exposure to 17-beta-estradiol had no effect. The tone-related release of NO was significantly increased after long-term exposure to 17-beta-estradiol. 6. In conclusion, this study indicate that the acute and long-term effects of 17-beta-estradiol in the rat aorta are mediated through different mechanisms. The long-term effect is mediated through the endothelium most likely by increasing NO release. In contrast, the acute effect of 17-beta-estradiol seems to be through an effect on the vascular smooth muscle cells.

Differential blockade of gamma-aminobutyric acid type A receptors by the neuroactive steroid dehydroepiandrosterone sulfate in posterior and intermediate pituitary.

Dehydroepiandrosterone sulfate (DHEAS) is a neuroactive steroid with antagonist action at gamma-aminobutyric acid type A (GABAA) receptors. Patch-clamp techniques were used to investigate DHEAS actions at GABAA receptors of the rat pituitary gland at two distinct loci: posterior pituitary nerve terminals and intermediate pituitary endocrine cells. The GABA responses in these two regions were quite different, with posterior pituitary responses having smaller amplitudes and desensitizing more rapidly and more completely. DHEAS blockade of GABAA receptors in the two regions also was different. In posterior pituitary, a site with an apparent dissociation constant of 15 microM accounted for most of the blockade, but a small fraction of blockade may be related to a site with a dissociation constant in the nanomolar range. In the intermediate lobe, DHEAS sensitivities in the nanomolar and micromolar ranges were clearly evident, in proportions that varied widely from cell to cell. Regardless of whether the GABA response of a cell was highly sensitive or weakly sensitive to DHEAS, GABA alone evoked currents that were indistinguishable in terms of amplitude, desensitization kinetics, and GABA sensitivity. Thus, the structural elements responsible for DHEAS blockade have a highly selective impact on receptor function. GABAA receptors with nanomolar sensitivity to DHEAS have not been described previously. This suggests that DHEAS may have an important role in the modulation of neuropeptide secretion, and the diverse properties of GABAA receptors in the rat pituitary provide mechanisms for selective regulation of the different peptidergic systems of this gland.

Effects of oral contraceptives on plasma neutral amino acids and cholesterol during a menstrual cycle.

OBJECTIVE: Concentrations of plasma neutral amino acids, i.e. threonine, serine, asparagine, glycine, alanine, citrulline, alpha-aminobutyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, and tryptophan, and serum cholesterol, were determined at the follicular (Day 4), mid-cycle (Day 16) and luteal (Day 25) phases of the menstrual cycle in 15 users of the new generation of combined oral contraceptives (OC), 11 on multiphase combined OC, and 17 controls. RESULTS: The controls showed a decrease in the sum of amino acids to 95% at mid-cycle and 90% in the luteal phase relative to the follicular phase, and a significant decrease in the tyrosine level at the luteal relative to the follicular phase. Since there was no significant difference between the two OC subgroups in the levels of the specified variables at either of the phases, the two groups were considered together. The sum of amino acids in the OC group decreased to 89% at mid-cycle and 91% at the luteal phase relative to the follicular phase, indicating less metabolic effect than reported for older OC formulations. Compared to the controls, the OC group showed significant increased threonine level at the luteal phase, decreased glycine levels at mid-cycle and the luteal phases, decreased citrulline level at mid-cycle, and markedly decreased tyrosine levels at the mid-cycle and luteal phases. Neither total nor high density lipoprotein (HDL) cholesterol differed significantly between the control and OC groups. CONCLUSION: The results suggest that the metabolic effects of the new generation combined OC on neutral amino acids and cholesterol are only modest to slight, except for the effect on tyrosine, the brain noradrenaline precursor, which may cause disturbances of various noradrenaline-mediated central functions in susceptible subjects.

PIP: Concentrations of plasma neutral amino acids and serum cholesterol were determined at the follicular (day 4), mid-cycle (day 16), and luteal (day 25) phases of the menstrual cycle in 15 users of the new generation of combined oral contraceptives (OCs), 11 on multiphase combined OCs, and 17 controls. The controls showed a decrease in the sum of amino acids to 95% at mid-cycle and 90% in the luteal phase relative to the follicular phase and a significant decrease in the tyrosine level in the luteal relative to the follicular phase. Since there was no significant difference between the two OC subgroups in the levels of the specified variables at either of the phases, the two groups were considered together. The sum of amino acids in the OC group decreased to 89% at mid-cycle and 91% in the luteal phase relative to the follicular phase, indicating less metabolic effect than reported for older OC formulations. Compared to the controls, the OC group showed significant increased threonine levels in the luteal phase, decreased glycine levels at mid-cycle and in the luteal phase, decreased citrulline levels at mid-cycle, and markedly decreased tyrosine levels at mid-cycle and in the luteal phase. Neither total nor high-density lipoprotein (HDL) cholesterol differed significantly between the control and OC groups. The results suggest that the metabolic effects of the new generation combined OCs on neutral amino acids and cholesterol are only modest to slight, except for the effect on tyrosine, the brain noradrenaline precursor, which may cause disturbances of various noradrenaline-mediated central functions in susceptible individuals.

Tyrosine metabolism in users of oral contraceptives.

Brain noradrenaline takes part in the regulation of several brain functions. The formation of brain noradrenaline depends on brain tyrosine (Tyr) levels, which associates with the ratio in plasma of Tyr to other large, neutral amino acids (LNAA). Tyr metabolism has been studied in users of the new generation combined oral contraceptives (OC) and comparable controls at the follicular, mid-cycle, and luteal phases of the menstrual cycle. OC users showed significantly increased plasma Tyr transaminase activity, and significantly decreased plasma Tyr and Tyr/LNAA levels at mid-cycle and luteal phase, whereas plasma total 3-methoxy-4-hydroxyphenylglycol (MHPG) was not affected. Following an oral protein load, the area under the curve in plasma of Tyr and Tyr/LNAA in OC users at the luteal phase were 43% and 29%, respectively, of control levels. The results suggest that the decreased Tyr availability to the brain in OC users may result in a substrate-limited reduction of brain noradrenaline formation, which, secondarily, may contribute to disturbances of mood, coping mechanisms, and appetite in susceptible subjects.

Dihydropyridine ligands influence the evoked release of oxytocin and vasopressin dependent on stimulation conditions.

The effects of dihydropyridine ligands on the electrically evoked release of neurohypophysial hormones from isolated, rat neurointermediate lobes were investigated as a function of all combinations of two pulse widths (0.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz). The dihydropyridine agonist (S)-(+)-202-791 potentiated concentration dependently the release of both oxytocin and vasopressin at a pulse width of 2 ms and a frequency of 6.5 Hz. This effect of (S)-(+)-202-791 was abolished by the antagonist (-)-nitrendipine and stereospecifically by (R)-(-)-202-791 (only vasopressin). The antagonist (R)-(-)-202-791 alone inhibited the release of oxytocin at 13 Hz and 2 ms. The results presented show that the effects of the dihydropyridine ligands are dependent on the stimulation conditions, and thus demonstrate that the entry of Ca2+ through the dihydropyridine sensitive L-type Ca2+ channel is associated with electrically evoked release of neurohypophysial hormones under certain conditions.

Ligand structural specificity of GABAA receptors in guinea pig ileum.

The ligand structural specificity of ileal GABAA receptors was examined using the strength and half-life of contractions in guinea-pig myenteric plexus-longitudinal muscle preparations. The agonists used differ by more than a factor of 1000 in affinity to central GABAA receptors and include both conformationally flexible and restricted molecules as well as pairs of enantiomers. The overall correlation between ileal contractile activity and rat brain receptor affinity was poor (r = 0.75), but within groups of conformationally flexible or conformationally restricted molecules a high correlation was found (r greater than 0.9999). When comparing data for ileal contractile activity with available data for agonist activity in the CNS no difference between ligand specificity of ileal and central GABAA receptors was apparent with the present range of ligands. The half-lives of ileal contractile responses were found to decrease with increasing GABAB agonist activity.

Influence of interleukin-1 beta on the secretion of oxytocin and vasopressin from the isolated rat neurohypophysis.

The effect of the cytokine interleukin-1 beta on the secretion of oxytocin and vasopressin from electrically stimulated rat neurohypophysis was examined in vitro. The release of oxytocin and vasopressin was concentration-dependently increased by interleukin-1 beta in the concentration range from 4.4 pM to 440 pM. The effect of interleukin-1 beta on oxytocin secretion was less intense as compared to vasopressin. After 440 pM interleukin-1 beta the electrically evoked release of oxytocin was increased about 22% and had not reached its maximum. The vasopressin response was maximal after 44 pM interleukin-1 beta, the response being increased 43% compared to control. No trace of interleukin-1 beta was found in the posterior pituitary (less than 350 pmol/lobe, radioimmunoassay). The results indicate that interleukin-1 beta might be involved the regulation of oxytocin and vasopressin at the pituitary level.

Kappa-opioid receptor agonists differentially affect the release of neurohypophysial hormones.

The influence of kappa-opioid receptor agonists and antagonists on release of oxytocin and vasopressin was examined in isolated rat neurointermediate lobes. Electrically evoked release of oxytocin and vasopressin was concentration-dependently inhibited by the specific kappa-receptor agonist U69593, whereas bremazocine only inhibited the secretion of oxytocin markedly. Treatment with naloxone enhanced the evoked release of oxytocin significantly without effect on vasopressin secretion. The U69593-mediated inhibition of oxytocin release was abolished by naloxone, whereas that of vasopression was unaffected. Naloxone did not reverse the bremazocine-induced inhibition of hormone release. The data support the theory of an inhibiting endogenous control over oxytocin secretion and show that the release of oxytocin and vasopresin is differentially affected by the two K-receptor agonists.

Interleukin-1 beta stimulates the release of vasopressin from rat neurohypophysis.

The release of vasopressin from the isolated superfused rat neurohypophysis was measured. The electrically evoked release of vasopressin after phasic submaximal stimulation was increased on exposure to the cytokine, interleukin-1 beta (44 pM). The release returned to its control level when the peptide was withdrawn. The results indicates a permissive role of interleukin-1 beta in the release of vasopressin.

Biphasic effect of a kappa-opioid receptor agonist on plasma oxytocin levels in rats.

The effect of the kappa-opioid receptor agonist, bremazocine, on plasma oxytocin levels in rats was measured by a sensitive radioimmunoassay. Initially, a decrease in plasma oxytocin levels was seen 30 min after injection. This was in accordance with the bremazocine inhibition of oxytocin release after submaximal electrical stimulation seen in isolated neurointermediate lobes. The initial decrease in plasma oxytocin reversed, and 4 h after injection of bremazocine a 20-fold increase in the oxytocin level was seen. The rise in plasma oxytocin was paralleled by a rise in plasma sodium. The biphasic time course of the plasma oxytocin response can be explained by a combination of an inhibition of oxytocin release from the neurohypophysis and an increased water excretion leading to an elevation in plasma sodium, which may be responsible for the late rise in plasma oxytocin. Down-regulation of the opioid receptors may also contribute to the delayed rise in plasma oxytocin.

The influence of morphine and naloxone on plasma oxytocin concentration in the rat.

Subcutaneous injections of morphine decrease the plasma oxytocin level significantly in both normohydrated rats and rats dehydrated for 24 hrs. This effect of morphine is naloxone-reversible. Injections of naloxone increase the plasma oxytocin level in a time dependent manner, when the oxytocin system is stimulated by dehydration, but have no effect on basal oxytocin level. This observation indicates liberation of an endogenous opioid, during stimulation of the neurohypophysis, leading to inhibition of the oxytocin secretion. A discrepancy in the time course between the plasma oxytocin enhancing effect and the morphine reversing effect of naloxone is observed. On the basis of this observation two different opioid receptors are suggested to be involved in the effect of naloxone on the neurohypophysis.

Role of gastrin-releasing peptide in pepsinogen secretion from the isolated perfused rat stomach.

We studied the effects of the neuropeptide gastrin-releasing peptide on pepsinogen secretion using an isolated perfused rat stomach with intact vagal innervation. Following electrical stimulation of the vagus nerves, the pepsin output to the luminal effluent increased from 94 +/- 7 to 182 +/- 24 units pepsin/min and the release of immunoreactive gastrin-releasing peptide to the venous effluent increased from 0.059 +/- 0.014 to 0.138 +/- 0.028 pmol/min. Infusion of gastrin-releasing peptide at 10(-8) M significantly increased pepsin output (from 87 +/- 17 to 129 +/- 22 units pepsin/min) and simultaneous infusion of gastrin-releasing peptide and carbachol at 10(-8) and 10(-6) M, respectively, resulted in an increase to almost 4 times the basal values. Atropine reduced but did not abolish the pepsin response to vagal stimulation and to infusion of gastrin-releasing peptide. Our results suggest that gastrin-releasing peptide participates in the vagal control of pepsinogen secretion.

GABA receptor stimulation increases the release of vasopressin and oxytocin in vitro.

The release of oxytocin and vasopressin from rat neurointermediate lobes was determined in vitro. The electrically evoked release of posterior pituitary hormones was markedly potentiated by the GABA receptor agonist, isogauvacin, an effect which was abolished by the GABAA receptor antagonist bicuculline. Spontaneous hormone outflow was not affected by the substances tested. The results suggest the existence of a GABA receptor on the terminal fibres in the pituitary, facilitating the release of oxytocin and vasopressin.

Pharmacological profile of a novel class of muscarinic acetylcholine receptor agonists.

Some in vivo pharmacological effects of a number of muscarinic acetylcholine receptor agonists containing the bicyclic 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) skeleton were studied in rats and mice. The key compounds O,N-dimethyl-THPO (2) and O-methyl-THPO (4) are bioisosteres of arecoline and norarecoline, respectively. The vasodepressor effects of arecoline and the title compounds in anaesthetized rats gave parallel log dose-response curves. The order of potency of the compounds in this test system was identical with that measured earlier using a guinea-pig ileum preparation, arecoline and 2 being the most active compounds. This order of potency was different from those for the antinociceptive and anticonvulsant effects of the compounds using grid shock and isoniazid antagonism tests, respectively, where O-propargyl-THPO (3) proved to be the most active. The pA2 values for the atropine or scopolamine antagonism of these effects of arecoline and 4 were calculated. The partition coefficients (log P values) of the compounds were measured and shown to conform with their ability to penetrate the blood-brain barrier.

Morphine antidiuresis in conscious rats: contribution of vasopressin and blood pressure.

Intracerebroventricular injections of morphine (5-20 micrograms) produced a dose dependent antidiuresis in the conscious hydrated rat. Naloxone pretreatment completely abolished this antidiuretic effect, but pretreatment with a specific antidiuretic vasopressin antagonist did not change the morphine antidiuresis. The vasopressin concentration in the first voided urine after antidiuresis from morphine treated rats was found to be in the same range as the vasopressin concentration in urine from saline treated rats. Injections of 20 micrograms morphine intracerebroventricularly in conscious hydrated rats gave a short decrease in heart rate but not in mean arterial blood pressure. This indicates, that either liberation of vasopressin nor a fall in systemic blood pressure contribute to the morphine antidiuresis.

Extremely decreased release of prostaglandin E2-like activity from chopped lung of ethyl linolenate-supplemented rats.

Three groups of weanling male rats were reared on a fat-free diet for 13 weeks. One group received only the fat-free diet (FF rats), the other 2 groups received the fat-free diet and a daily supplement of 2 energy% ethyl linoleate ([n-6] rats), or 2 energy% ethyl linolenate ([n-3] rats). The chopped lung preparation was used to illustrate an in vitro prostaglandin formation. PGE2-like activity was quantified on rat stomach strip. The release of PGE2-like activity expressed as ng PGE2-equivalent per g lung tissue (mean +/- SD) was 23 +/- 7, less than 6, and 65 +/- 20 for the FF rats, the (n-3) rats, and the (n-6) rats, respectively. PGE2 quantification by radioimmunoassay of the chopped lung effluent collected after passing over the rat stomach strip revealed the same release pattern as the bioassay. Fractionation of chopped lung effluent on HPLC with radioimmunoassay detection indicated that the lung tissue from (n-3) rats released very little PGE3, if any, in spite of a 20:5(n-3)/20:4(n-6) ratio of 5.2 in the lipids of the lung. It is suggested that the pool of arachidonic acid for prostaglandin production in vitro is different from the one which functions in vivo, and the these pools are differently affected by dietary EFA.

Coronary vasodilatory, spasmolytic and cAMP-phosphodiesterase inhibitory properties of dihydropyranocoumarins and dihydrofuranocoumarins.

Twenty-three dihydropyrano- and dihydrofuranocoumarins, most of plant origin, were examined for their effects on the coronary flow of isolated perfused guinea-pig heart, on the Ba2+-induced spasms in isolated guinea-pig ileum, on the cAMP level in guinea-pig heart homogenate and on the cAMP metabolising activity of purified beef heart cAMP-phosphodiesterase. For certain esters of dihydropyranocoumarin- and dihydrofuranocoumarin alcohols coronary vasodilatory and spasmolytic activities comparable to those of papaverine were observed. A very close correlation between the coronary vasodilatory and the spasmolytic activity was found. The most potent structures maximally increased the cAMP level from 19 pmol/mg protein to about 60 pmol/mg protein and inhibited the cAMP-phosphodiesterase activity with about 90%. The potencies were comparable to those of papaverine. A significant correlation was obtained between the coronary vasodilatory and the cAMP-phosphodiesterase inhibitory activity. The results indicate involvement of cAMP-phosphodiesterase inhibition in coronary vasodilatory effects of acyloxydihydropyrano- and acyloxydihydrofurano-coumarins.