Site-specific effects of the nonsteroidal anti-inflammatory drug lysine clonixinate on rat brain opioid receptors.

In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 x 10(-4) mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

Antispasmodic action of propinox on the isolated human gallbladder: possible mechanism of action.

Propinox is an antispasmodic drug frequently used in the treatment of disorders of the gastrointestinal tract, the uterus and the gallbladder, but little is known about its relaxing activity in gallbladder tissue. The main objective of this study was to determine the antispasmodic activity of propinox, compared to other antispasmodics, in the gallbladder and to assess its binding affinity to receptor sites which may be involved in its mechanism of action. Antispasmodic activity of propinox, (-) scopolamine-n-butyl bromide, atropine and verapamil was determined in human gallbladders to reduce the risk of interspecies variability. Inhibitory activities (ED50) of carbachol-induced contraction were: atropine 5.03 x 10(-8) M > propinox 1.25 x 10(-7) M > verapamil 6.63 x 10(-6) M > (-) scopolamine-n-butyl bromide 5.4 x 10(-5) M. pD'2 for propinox was 6.94, indicating non competitive inhibition of carbachol action. Radioligand binding studies were performed to determine if the antispasmodic action of the drug involved binding to muscarinic receptors or calciumantagonist sites. The inhibition constant (Ki) of propinox for muscarinic receptors of guinea pig ileum smooth muscle, which contains a mixed M2-M3 receptor population, was 1.6 x 10(-6) M. Ki for brain muscarinic receptors (M1) was 1.0 x 10(-4) M, for cardiac receptors (M2) 1.2 x 10(-6) M and from salivary gland receptors (M3) 1.5 x 10(-6) M. For binding to the dihidropiridine calcium antagonist binding sites, Ki were: 4.9 x 10(-5) M for propinox and 2.2 x 10(-7) M for verapamil. For the phenylalkylamine binding sites Ki were: 5.0 x 10(-6) M for propinox and 3.5 x 10(-8) M for verapamil. For the benzothiacepine binding sites, Ki for propinox was 5.2 x 10(-6) M. The following may be concluded: 1.--The antispasmodic activity of propinox in isolated human gallbladder was comparatively less potent than that of atropine and more potent than those of verapamil and (-) scopolamine-n-butyl bromide. 2.--Propinox showed binding to muscarinic and calcium receptors that can be related to its antispasmodic activity; suggesting that the drug is an antispasmodic with anticholinergic and musculotropic activity. 3.--The dual mechanism of action, anticholinergic and calcium-blocking, would induce synergism of pharmacodynamic effects and minimize adverse events of pure antimuscarinic drugs or calcium antagonists.

Antispasmodic action of propinox on the isolated human gallbladder: possible mechanism of action

Propinox is an antispasmodic drug frequently used in the treatment of disorders of the gastrointestinal tract, the uterus and the galbladder, but little is known about its relaxing activity in gallbladder tissue. The main objective of this study was to determine the antispasmodic activity of propinox, compared to other antispasmodics, in the gallblader and to assess its binding affinity to receptor sites which may be involved in its mechanism of action. Antispasmodic activity of propinox, (-) scopolamine-n-butyl bromide, atropine and verapamil was determined in human gallbladders to reduce the risk of interspecies variability. Inhibitory activities (ED50) of carbachol-induced contraction were: atropine 5.03x10(-8) M>propinox 1.25x10(-7) M> verapamil 6.63x10(-6)M> (-) scopolamine-n-butyl1 bromide 5.4x10(-5) M. pD'2 for propinox was 6.94, indicating non competitive inhibition of carbachol action. Radioligand binding studies were performed to determine if the antisplasmodic action of the drug involved binding to muscarinic receptors or calciumatagonist sites. The inhibition constant (Ki) of proponix for muscarinic receptors of guinea pig ileum smoth muscle, which contains a mixed M2-M3 receptor population, was 1.6x10(-6) M. Ki for brain muscarinic receptors (M1) was 1.0x10(-4) M, for cardiac receptors (M1) was 1.0x10(-4)M, for receptors (M2) 1.2x10(-6)M and from salivary gland receptors (M3) 1.5x10(-6)M. For binding to the dihidropiridine calcium antagonist binding sites, Ki were: 4.9x10(-5)M for propinox and 2.2x10(-7)M for verapamil. For the phenylakylamine binding sites Ki were: 5.0x10(-6)M for propinox and 3.5x10(-8)M for verapamil. For the benzothiacepine binding sites, Ki for propinox was 5.2x10(-6)M. The following may be concluded: 1- The antispasmodic activity of propinox in isolated human galbladder was was comparatively less potent than of atropine and more potent than those verapamil and (-) scopolamine-n-butyl bromide. 2- Propinox showed binding to muscarinic and calcium receptors that can be related to its antisplasmodic activity; suggesting that the drug is an antispasmodic with anticholinergic and musculotropic activity. 3.- The dual mechanism of action, anticholinergic and calcium-blocking, would induce synergism of pharmacodynamic effects and minimize adverse events of pure antimuscarinic drugs or calcium antagonists.

Antispasmodic action of propinox on the isolated human gallbladder: possible mechanism of action.

Propinox is an antispasmodic drug frequently used in the treatment of disorders of the gastrointestinal tract, the uterus and the gallbladder, but little is known about its relaxing activity in gallbladder tissue. The main objective of this study was to determine the antispasmodic activity of propinox, compared to other antispasmodics, in the gallbladder and to assess its binding affinity to receptor sites which may be involved in its mechanism of action. Antispasmodic activity of propinox, (-) scopolamine-n-butyl bromide, atropine and verapamil was determined in human gallbladders to reduce the risk of interspecies variability. Inhibitory activities (ED50) of carbachol-induced contraction were: atropine 5.03 x 10(-8) M > propinox 1.25 x 10(-7) M > verapamil 6.63 x 10(-6) M > (-) scopolamine-n-butyl bromide 5.4 x 10(-5) M. pD2 for propinox was 6.94, indicating non competitive inhibition of carbachol action. Radioligand binding studies were performed to determine if the antispasmodic action of the drug involved binding to muscarinic receptors or calciumantagonist sites. The inhibition constant (Ki) of propinox for muscarinic receptors of guinea pig ileum smooth muscle, which contains a mixed M2-M3 receptor population, was 1.6 x 10(-6) M. Ki for brain muscarinic receptors (M1) was 1.0 x 10(-4) M, for cardiac receptors (M2) 1.2 x 10(-6) M and from salivary gland receptors (M3) 1.5 x 10(-6) M. For binding to the dihidropiridine calcium antagonist binding sites, Ki were: 4.9 x 10(-5) M for propinox and 2.2 x 10(-7) M for verapamil. For the phenylalkylamine binding sites Ki were: 5.0 x 10(-6) M for propinox and 3.5 x 10(-8) M for verapamil. For the benzothiacepine binding sites, Ki for propinox was 5.2 x 10(-6) M. The following may be concluded: 1.--The antispasmodic activity of propinox in isolated human gallbladder was comparatively less potent than that of atropine and more potent than those of verapamil and (-) scopolamine-n-butyl bromide. 2.--Propinox showed binding to muscarinic and calcium receptors that can be related to its antispasmodic activity; suggesting that the drug is an antispasmodic with anticholinergic and musculotropic activity. 3.--The dual mechanism of action, anticholinergic and calcium-blocking, would induce synergism of pharmacodynamic effects and minimize adverse events of pure antimuscarinic drugs or calcium antagonists.

Antispasmodic action of propinox on the isolated human gallbladder: possible mechanism of action

Propinox is an antispasmodic drug frequently used in the treatment of disorders of the gastrointestinal tract, the uterus and the galbladder, but little is known about its relaxing activity in gallbladder tissue. The main objective of this study was to determine the antispasmodic activity of propinox, compared to other antispasmodics, in the gallblader and to assess its binding affinity to receptor sites which may be involved in its mechanism of action. Antispasmodic activity of propinox, (-) scopolamine-n-butyl bromide, atropine and verapamil was determined in human gallbladders to reduce the risk of interspecies variability. Inhibitory activities (ED50) of carbachol-induced contraction were: atropine 5.03x10(-8) M>propinox 1.25x10(-7) M> verapamil 6.63x10(-6)M> (-) scopolamine-n-butyl1 bromide 5.4x10(-5) M. pD2 for propinox was 6.94, indicating non competitive inhibition of carbachol action. Radioligand binding studies were performed to determine if the antisplasmodic action of the drug involved binding to muscarinic receptors or calciumatagonist sites. The inhibition constant (Ki) of proponix for muscarinic receptors of guinea pig ileum smoth muscle, which contains a mixed M2-M3 receptor population, was 1.6x10(-6) M. Ki for brain muscarinic receptors (M1) was 1.0x10(-4) M, for cardiac receptors (M1) was 1.0x10(-4)M, for receptors (M2) 1.2x10(-6)M and from salivary gland receptors (M3) 1.5x10(-6)M. For binding to the dihidropiridine calcium antagonist binding sites, Ki were: 4.9x10(-5)M for propinox and 2.2x10(-7)M for verapamil. For the phenylakylamine binding sites Ki were: 5.0x10(-6)M for propinox and 3.5x10(-8)M for verapamil. For the benzothiacepine binding sites, Ki for propinox was 5.2x10(-6)M. The following may be concluded: 1- The antispasmodic activity of propinox in isolated human galbladder was was comparatively less potent than of atropine and more potent than those verapamil and (-) scopolamine-n-butyl bromide. 2- Propinox showed binding to muscarinic and calcium receptors that can be related to its antisplasmodic activity; suggesting that the drug is an antispasmodic with anticholinergic and musculotropic activity. 3.- The dual mechanism of action, anticholinergic and calcium-blocking, would induce synergism of pharmacodynamic effects and minimize adverse events of pure antimuscarinic drugs or calcium antagonists. (AU)

Adrenocorticoid action in the spinal cord: some unique molecular properties of glucocorticoid receptors.

1. Glucocorticoid hormones affect several functions of the spinal cord, such as synaptic transmission, biogenic amine content, lipid metabolism, and the activity of some enzymes (ornithine decarboxylase, glycerolphosphate dehydrogenase), indicating that this tissue is a target of adrenal hormones. 2. Corticosterone, the main glucocorticoid of the rat, is detected at all regional levels of the spinal cord, and cold stress increases this steroid, predominantly in the cervical regions. 3. Intracellular glucocorticoid receptors have been found in the spinal cord, with higher concentrations in the cervical and lumbar enlargements. Prima facie, these receptors presented biochemical, stereospecifical, and physicochemical properties similar to those of receptors found in other regions of the nervous system. The prevalent form in the spinal cord is the type II receptor, although type I is also present in small amounts. 4. The type II glucocorticoid receptor of the spinal cord shows an affinity lower (Kd 3.5 nM) than that of the hippocampal type II site (Kd 0.7 nM) when incubated with [3H]dexamethasone. This condition may impair the nuclear translocation of the spinal cord receptor. 5. Another peculiar property of spinal cord type II site is a greater affinity for DNA-cellulose binding than the hippocampal receptor during heat-induced transformation. Also, the spinal cord receptor shows resistance to the action of RNAse A, an enzyme which increases DNA-cellulose binding of the hippocampal receptor, indicating that both receptors may be structurally different. 6. Therefore, it is possible that a different subclass of type II, or "classical glucocorticoid receptor," is present in the spinal cord. This possibility makes the cord a useful system for studying diversity of glucocorticoid receptors of the nervous system, especially the relationship between receptor structure and function.

Heterogeneity and properties of transformation of corticosteroid receptors in spinal cord and hippocampus.

The central nervous system contains two classes of corticoid receptors, named types I and II following terminology accepted for the kidney. Phenotypically, type I sites are differentiated into a corticosterone (CORT)-preferring species (Ia) and a mineralocorticoid receptor (Ib). These populations were tentatively compared in the spinal cord and hippocampus. Using [3H]dexamethasone (DEX) and selective blockage of sites, we have observed that type II receptors were comparable in both tissues, while Ia was almost exclusive of the hippocampus. Saturation analysis using [3H]DEX demonstrated that type Ia was a low affinity receptor (Kd approximately equal to 2-5 nM) while type II was a higher affinity site (KdII less than KdI). Using [3H]CORT, or [3H]aldosterone (ALDO), as ligand, preferential labeling of type I sites was achieved, always showing higher concentrations in the hippocampus. Therefore, [3H]DEX seems a ligand of choice to visualize types Ia and II receptors. Another difference noted between the spinal cord and hippocampus, pertained to the sensitivity towards the enzyme RNAse A, which increases heat-induced transformation of the bound receptor, according to the results of DNA cellulose affinity chromatography. In these experiments, type I sites of both spinal cord and hippocampus, plus type II of hippocampus, showed sensitivity toward the enzyme, whereas type II of the spinal cord was refractory to RNAse A enhancement of transformation. These results indicate that the dynamics of transformation is different among receptors showing similar affinity and competition, suggesting further heterogeneity due to receptors themselves, or to tissue factors regulating their biochemical properties.

Steps of glucocorticoid action in normal and diabetic rat placenta.

This investigation examined the effects of Streptozotocin diabetes in pregnancy on several parameters of glucocorticoid action in the rat placenta. Pregnant diabetic rats showed reduced body weight, increased adrenal weight and serum corticosterone concentrations. Glucocorticoid receptors in placental cytosol of labyrinthine zone, measured in the absence of MoO4Na2 were similar in control and diabetic rats, but after addition of MoO4Na2 receptor number were moderately, but significantly reduced in diabetic placentas (P less than 0.01). No changes in affinity were detected in saturation analysis. Furthermore, transformation of the receptor assessed by its capacity for binding to DNA-cellulose, was enhanced in diabetic animals, suggesting increased efficiency of the receptor-bound hormone. Since the function of the glucocorticoid receptor of rat placenta may be the inhibition of local progesterone production (Heller and De Nicola, J. steroid Biochem. 19 (1983) 1339-1343), we determined progesterone synthesis in vitro and found that diabetic placentas synthesized significantly less progesterone than control tissue (P less than 0.05). Lastly, we found that the metabolism of corticosterone to 11-dehydrocorticosterone, while declining in control placentas as pregnancy advanced, it was sustained in diabetic pregnancy. It is suggested that diabetic rat placentas showed increased activity towards the glucocorticoid receptor, resulting in reduction in progesterone synthesis and sustained catabolism of corticosterone. The latter may possibly constitute a compensatory mechanism to protect the fetal compartment from high levels of maternal glucocorticoids.

Functions and dysfunctions of receptors for adrenal corticoids in the central nervous system.

Glucocorticoids (GC) have several known effects on the function of the nervous system, and GC receptors have been identified in regions responding to hormonal action. In the spinal cord, GC receptors have been characterized in vitro, which share several biochemical properties in common with receptors in better studied areas such as the hippocampus. Moreover, enzymes which are induced by GC in the hippocampus, such as glycerolphosphate dehydrogenase and ornithine decarboxylase, are also under specific GC control in the spinal cord. Yet GC receptors in the latter tissue divert from those in hippocampus during some in vivo as well as in vitro studies. In vivo, uptake of [3H]corticosterone by purified cell nuclei was 5-8-fold higher in the hippocampus as compared to the cord. In vitro, a higher percentage of GC receptors previously transformed by heating, showed affinity towards DNA-cellulose in the spinal cord than in the hippocampus. The enzyme RNAse A effectively increased receptor binding to DNA-cellulose in hippocampus, whereas the cord was insensitive to its action. These results suggest that there is a "receptor dysfunction" in the spinal cord, the significance of which is poorly understood in terms of the accepted model of steroid hormone action.

Hormonal effects on unoccupied estrogen receptors in nuclei of anterior pituitary glands.

Free nuclear receptors were measured in anterior pituitaries from ovariectomized-adrenalectomized rats after extraction of purified nuclei with 0.4 M KC1 and incubation of the extract with 2.5 nM (3H)-estradiol (E2) during 1.5 h at 0-4 degrees C. High affinity, low capacity binding was present in untreated rats; receptor concentration doubled after E2 was given for 4 days, whereas acute (60 min) treatment was without effect. Prolonged exposure to diethylstilbestrol (3 months) down-regulated free as well as total nuclear receptors. Tamoxifen increased free nuclear receptors, induced the progestin receptor in cytosol, and inhibited E2-stimulated serum prolactin but not E2 stimulation of free nuclear receptors. Progesterone and testosterone had no effect on basal or E2-stimulated free nuclear sites, whereas dexamethasone reduced the former but not the latter. Furthermore, from 33 to 36% of free nuclear receptors bound to (3H)-E2, were retained in DNA-cellulose columns whether incubation with ligand was performed at low (0-4 degrees C) or high temperature (25 degrees C); DNA-cellulose binding was unchanged after short E2 but increased after 4 days of E2. These data suggest ligand as well as antiestrogen and corticoid regulation of free nuclear receptors for E2 in anterior pituitary. Free nuclear sites may be a fraction of total cell receptors which are in the transformed state and which bind more tightly to nuclei in vivo, as indicated by the results of DNA-cellulose binding in vitro.

Hormonal effects on unoccupied estrogen receptors in nuclei of anterior pituitary glands.

Free nuclear receptors were measured in anterior pituitaries from ovariectomized-adrenalectomized rats after extraction of purified nuclei with 0.4 M KC1 and incubation of the extract with 2.5 nM (3H)-estradiol (E2) during 1.5 h at 0-4 degrees C. High affinity, low capacity binding was present in untreated rats; receptor concentration doubled after E2 was given for 4 days, whereas acute (60 min) treatment was without effect. Prolonged exposure to diethylstilbestrol (3 months) down-regulated free as well as total nuclear receptors. Tamoxifen increased free nuclear receptors, induced the progestin receptor in cytosol, and inhibited E2-stimulated serum prolactin but not E2 stimulation of free nuclear receptors. Progesterone and testosterone had no effect on basal or E2-stimulated free nuclear sites, whereas dexamethasone reduced the former but not the latter. Furthermore, from 33 to 36

of free nuclear receptors bound to (3H)-E2, were retained in DNA-cellulose columns whether incubation with ligand was performed at low (0-4 degrees C) or high temperature (25 degrees C); DNA-cellulose binding was unchanged after short E2 but increased after 4 days of E2. These data suggest ligand as well as antiestrogen and corticoid regulation of free nuclear receptors for E2 in anterior pituitary. Free nuclear sites may be a fraction of total cell receptors which are in the transformed state and which bind more tightly to nuclei in vivo, as indicated by the results of DNA-cellulose binding in vitro.