β-adrenoceptor stimulation-induced increase in the number of α2-adrenoceptors of cerebral cortical membranes in rats

Effects of pre-treatment of synaptic membranes with β-adrenoceptor agonists and cholera toxin on [³H]clonidine and [³H]yohimbine binding were examined in rat cerebral cortex. Pre-incubation of cerebral cortical membranes with isoproterenol (10 or 200 μM) or dobutamine (1, 10 or 200 μM) at 37 °C for 40 min caused a significant elevation of specific [³H]clonidine binding but treatment with salbutamol (10 or 200 μM) did not. Scatchard analysis showed that 200 μM isoproterenol treatment resulted in a significant elevation of high affinity component of [³H]clonidine binding which was significantly decreased by the addition of 10μM GTP. A significant elevation in high affinity [³H]clonidine binding was observed by treatment with 100 μg/ml cholera toxin, while a significant decrease in low affinity one was by the treatment. Specific [³H]yohimbine binding was also elevated by 10 or 200 μM isoproterenol treatment. It is suggested that stimulation of β-receptors, presumably β₁-subtype could elevate the number of agonist and antagonist binding sites in α₂-receptors in synaptic membranes by partially mediated by stimulatory and/or inhibitory GTP binding regulatory proteins.     

Interaction between alpha 2- and beta-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for beta-adrenergic receptors.

The interaction between alpha 2- and beta-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [3H]dihydroalprenolol ([3H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 microM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 microM clonidine, the maximum concentration which did not inhibit [3H]DHA binding, inhibition curves of [3H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 microM clonidine decreased a beta-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 microM yohimbine completely prevented the clonidine-induced reduction in the beta-adrenergic high-affinity state. In the presence of 200 microM GTP, the effect of clonidine was not observed. In addition, Kd and Bmax values for [3H]p-aminoclonidine ([3H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [3H]PAC binding. Moreover, isoproterenol inhibition of [3H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 microM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for beta-adrenergic receptors, while isoproterenol did not modulate the binding characteristics of alpha 2-adrenergic receptors. Furthermore, these results suggest that regulation between alpha 2- and beta-adrenergic receptors is not bidirectional, but is instead unidirectional from alpha 2-adrenergic receptors to beta-adrenergic receptors.     

Alpha 2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [3H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither Kd nor Bmax values in [3H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in alpha 2-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catalytic units decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.     

The interaction between β- and α2-adrenoceptors in cerebral cortical membranes: Isoproterenol-induced increase in [H]clonidine binding in rats

The pretreatment of rat cerebral cortical membranes with 10 or 100 μM isoproterenol at 37°C for 40 min caused a significant elevation of the B_max value of [³H]clonidine binding but pretreatment at 4°C did not affect the value. The isoproterenol-induced increase in the B_max value of the binding was higher in 50 mM Tris-HCl buffer (pH 7.7) than in Krebs-Ringer solution. In 50 mM Tris-HCl buffer (pH 7.7), treatment with isoproterenol reduced the B_max value of [³H]dihydroalprenolol binding but neitherK_d nor B_max of [³H]WB 4101 binding was affected by this treatment. Fitty μM propranolol or 100 μM GTP produced a significant reduction in isoproterenol-induced elevation of the B_max value of [³H]clonidine binding. In contrast, 100 μM cyclic AMP did not affect the control binding and 0.1 or 1 mM theophylline did not affect the isoproterenol-induced elevation of the binding. The only B_max value in high affinity binding of [³H]clonidine was increased by isoproterenol.It is suggested that isoproterenol increases the density of α₂-adrenoceptors in a temperature-dependent manner. The direct interaction between β- and α₂-receptor molecules and/or their indirect interaction, mediated by GTP regulatory proteins, would exist in the cerebral cortical membranes of rats.     

α2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [³H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither K_d nor B_max values in [³H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in α₂-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catylytic unit decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.     

Interaction betweenα2- and β-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for β-adrenergic receptors

The interaction betweenα₂- and β-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [³H]dihydroalprenolol ([³H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 μM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 μM clonidine, the maximum concentration which did not inhibit [³H]DHA binding, inhibition curves of [³H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 μM clonidine decreased a β-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 μM yohimbine completely prevented the clonidine-induced reduction in the β-adrenergic high-affinity state. In the presence of 200 μM GTP, the effect of clonidine was not observed. In addition,K_d andB_max values for[³H]p-aminoclonidine ([³H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [³H]PAC binding. Moreover, isoproterenol inhibition of [³H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 μM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for β-adrenergic receptors, while isoproterenol did not modulate the binding characteristics ofα₂-adrenergic receptors. Furthermore, these results suggest that regulation betweenα₂- and β-adrenergic receptors is not bidirectional, but is instead unidirectional fromα₂-adrenergic receptors to β-adrenergic receptors.     

Characterization of [3H]rauwolscine binding to alpha 2-adrenoceptor sites in the lumbar spinal cord of the cat: comparison to such binding sites in the cat frontal cerebral cortex

The binding of the selective alpha 2-adrenoceptor antagonist radioligand [3H]rauwolscine ([3H]RAUW) to homogenates of cat frontal cerebral cortex and cat lumbar spinal cord was investigated. Experiments were performed at 20 degrees C in 50 mM Tris HCl/l mM Na2EDTA buffer (pH 6.9 at 20 degrees C). At this temperature, specific [3H]RAUW binding, defined as the difference between the amount of [3H]RAUW bound in the absence and presence of 1 microM rauwolscine or 1 microM rauwolscine or 1 microM yohimbine, reaches equilibrium values by approximately 60 min and is reversible with a mean t1/2 of dissociation of 15 min in cortex and 20 min in spinal cord. The kinetically determined Kd of [3H]RAUW (mean K-1/mean K1) was 0.59 nM and 1.68 nM in cortex and spinal cord, respectively. The results of equilibrium saturation experiments, routinely performed at [3H]RAUW concentrations between 0.1 nM and 6.0 nM, indicate that [3H]RAUW binds to saturable sites in both CNS regions of the cat. Scatchard plots of saturation isotherm data were consistently linear and the mean Kd value determined from 10 such experiments was 0.72 nM in frontal cortex and 0.82 nM in lumbar spinal cord. A mean Bmax value of 230 fmol/mg protein was determined for saturable [3H]RAUW binding sites in the cat frontal cortex. In teh cat lumbar spinal gray, a mean Bmax value for saturable [3H]RAUW binding sites of 75 fmol/mg protein was obtained. Saturable [3H]RAUW binding sites in the cat lumbar spinal gray are present at apparently equal density in dorsal and ventral horns. Inhibition experiments, performed at 0.2 nM or 0.4 nM [3H]RAUW, indicate that the binding sites labeled by [3H] RAUW possess a pharmacology characteristic of alpha-adrenoceptors. Thus, rauwolscine, yohimbine, and phentolamine compete for specific [3H]RAUW binding with high affinity and are much more potent inhibitors than corynanthine, prazosin, and propranolol. Mean Hill coefficients, calculated from logit-log plots of competition data, were close to one for all antagonists examined. L-Epinephrine and L-norepinephrine were 15-20 times more potent inhibitors of specific [3H]RAUW binding than were their corresponding D-isomers. The agonist inhibitor potency series: p-aminoclonidine = clonidine = L-epinephrine greater than L-norepinephrine much greater than isoproterenol, is that expected of alpha 2-adrenoceptor sites. Mean Hill coefficients efficients for all agonists were considerably less than one.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of platelet alpha 2 adrenoceptors and measurement in control and depressed subjects

The alpha-adrenoceptor on platelets has been characterized using 3H-yohimbine, 3H-dihydroergocriptine, and 3H-clonidine. The receptor, which exhibits the characteristics of an alpha 2-type, has a Bmax for dihydroergocriptine of 330 fmoles/mg protein, for yohimbine of 178 fmoles/mg protein, and for clonidine of 38 fmoles/mg protein. Clonidine, but not yohimbine binding, is decreased by the presence of K+, Na+, or Li+. Adenosine triphosphate (ATP) and the guanosine triphosphate (GTP) analogue, Gpp(NH)p, reduce the affinity of clonidine for its binding site. Acute exercise, such as playing squash, does not apparently alter the Bmax or Kd of 3H-yohimbine binding to platelet membranes, and in vitro studies, with intact platelets or platelet membranes, show that incubation with adrenalin (10 microM) does not induce alterations in Bmax or Kd. In the present study, no correlation was found between age and alpha 2-adrenoceptor numbers. There was no significant difference in the Bmax for 3H-yohimbine binding to platelet membranes from control and depressed subjects, although the mean value for the depressed group was some 10% lower than that for the corresponding control group. There were no overall significant and consistent effects of desipramine (DMI) treatment. After 2-3 days of treatment, the Bmax was reduced by 20%, after 7 days by 14%, and after 21 days it was 8% above the control value. When an additional group of patients on DMI (7 days of treatment) was analyzed using one supramaximal concentration of 3H-yohimbine, there was a significant decrease (25%) in binding.     

Long-term treatment with clonidine and alpha-receptors in the brain of normotensive rats

The aim of the present study was to investigate whether or not changes in rat brain alpha-adrenoceptors take place during chronic treatment with a low dose of clonidine. Male Wistar normotensive rats were treated with clonidine (0.1 mg/kg)i.p. twice daily for 12 days. This treatment caused a significant increase in [3H]clonidine and in [3H]WB4101 binding, respectively, to alpha 2- and to alpha 1-adrenoceptors of the frontal cortex; the levels were 30% for [3H]clonidine and 20% for [3H]WB4101. The Scatchard analysis of data obtained in binding studies indicated that the enhanced binding of two ligands to membranes prepared from chronically clonidine-treated animals, was due to an apparent increase in the number of binding sites. These changes were seen 4 h after administration of the last treatment, before the appearance of the withdrawal syndrome. However, noradrenergic alpha 2-autoreceptors of synaptosomes, from the frontal cortex and hypothalamus of treated animals, were sensitive to the regulatory action of clonidine or of noradrenaline on the [3H]noradrenaline overflow elicited by high K+ as well as on the control animals. On the contrary, the alpha 2-receptors on the serotoninergic nerve terminals from the frontal cortex of treated animals were more sensitive than those of control animals to the action of clonidine or of noradrenaline in counteracting the [3H]5-hydroxytryptamine overflow elicited by high K+. These results suggest that during treatment with clonidine no autoreceptor hyposensitivity to the regulatory action of clonidine or noradrenaline on [3H]noradrenaline overflow elicited by high K+ takes place, but, as a consequence of the diminished noradrenaline availability at the synaptic cleft, the binding of [3H]WBA101 to alpha 1-receptors and of [3H]clonidine to pre- and postsynaptic alpha 2-receptors were significantly elevated in the frontal cortex, a brain areas where the alpha-2-receptors are mainly postsynaptic. Thus, the neurotransmitter concentration in the synaptic cleft may be responsible for the trans-synaptic modulation of the alpha 2-adrenoceptor postsynaptic population. In fact, the alpha 2-adrenoceptors which are presynaptically located on the serotoninergic terminals, but are postsynaptic in relation to the noradrenergic neurons, also show increased sensitivity after chronic clonidine treatment.     

Coupling of human brain cerebral cortical alpha 2-adrenoceptors to GTP-binding proteins in Alzheimer's disease.

The coupling of alpha 2-adrenoceptors to guanine nucleotide binding G-proteins was investigated in cerebral cortical membranes from control and Alzheimer's disease brain by characterizing the effects of MnCl2 and Gpp[NH]p on [3H]clonidine binding. The manganese induced elevation of [3H]clonidine binding was apparent in both control and Alzheimer's disease samples and the effect showed no significant difference between the two groups in the frontal cortex. However, the MnCl2 concentration dependence curves for control and Alzheimer's disease samples were significantly different from one another in the temporal cortex, whereas the pattern of stimulation by MnCl2 remained the same. The guanine nucleotide analogue, Gpp[NH]p inhibited [3H]clonidine binding in a concentration-dependent manner, the profiles of inhibition showing no significant differences between control and Alzheimer's disease samples. Analysis of the effect of Gpp[NH]p on [3H]clonidine saturation binding curves showed no significant differences between control and Alzheimer's disease samples in either frontal (Kd = 9.68 +/- 1.38, 9.1 +/- 2.6 nM; Bmax = 40.23 +/- 4.33, 44.3 +/- 9.4 fmol/mg, control and Alzheimer's disease values, respectively), or temporal (Kd = 11.61 +/- 4.04, 5.38 +/- 2.5 nM; Bmax = 52.0 +/- 14.0, 31.07 +/- 8.00 fmol/mg control and Alzheimer's disease values, respectively) cortices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Idazoxan and brain α2-adrenoceptors in the rabbit

The effect of intravenous infusion of idazoxan on the depressor response to intracisternal clonidine 1 microgram/kg and on [3H]yohimbine binding in the fore- and hindbrain of the rabbit was examined. Idazoxan was infused either acutely (30 min) or chronically (5 days) at doses of 0.56 or 1.1 mg/h. Idazoxan 1.1 mg/h reduced the fall in blood pressure after clonidine. This attenuation of the depressor response was observed in the groups that were given the higher dose of idazoxan both acutely and chronically. The extent of attenuation was not modified by the duration of treatment. The low dose of idazoxan given acutely had no significant effect on the response to clonidine but the chronically infused group showed an enhanced response. A significant increase in the number of [3H]yohimbine binding sites (83%) was observed in the forebrain after 5 days infusion of 1.1 mg/h idazoxan with no change in the hindbrain. The lower dose of infusion did not cause any significant change in [3H]yohimbine binding in either brain region. Thus it appears that the susceptibility of the alpha 2-adrenoceptor binding sites to up-regulation by idazoxan may depend on the brain region observed.     

Alpha 1-adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [125I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70-85% specific. Various adrenergic agonists and antagonists competed for [125I]HEAT binding according to the potencies of prazosin greater than, yohimbine greater than or equal to, clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showed the Bmax of 209 fmol/mg protein and a Kd of 184 pM for [125I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [125I]HEAT binding sites with a maximal response observed after 8 h at 100 microM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 microM NE. Clonidine expressed differential effects on alpha 1-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 microM clonidine caused a 40% decrease in the Bmax of [125I]HEAT binding without influencing the Kd value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [125I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

Attenuation by chronic imipramine treatment of [H]clonidine binding to cortical membranes and of clonidine-induced hypothermia: the influence of central chemosympathectomy

Central chemosympathectomy with intraventricular injection of 250 μg of 6-hydroxydopamine, resulting in depression of cerebral noradrenaline level by 75%, led to an increase in specific binding of [³H]clonidine to cerebral cortical membranes, but did not affect the hypothermic response to clonidine. Chronic imipramine treatment depressed [³H]clonidine binding and attenuated clonidine-induced hypothermia similarly in non-chemosympathectomized and chemosympathectomized rats.     

Spinal nerve ligation increases alpha2-adrenergic receptor G-protein coupling in the spinal cord

Intrathecal and epidural administration of the alpha2-adrenergic receptor agonist clonidine in humans results in analgesia to both acute nociceptive and chronic neuropathic pain. The potency of clonidine increases with hypersensitivity to mechanical stimuli after nerve injury, although the reasons for this change are unknown. In the present study, we tested the hypothesis that peripheral nerve injury alters either spinal alpha2-adrenergic receptor-mediated G-protein activity or alpha2-adrenergic receptor number. Rats were randomized to left spinal nerve ligation (SNL) or sham surgery. Tactile hypersensitivity in the hindpaw was confirmed and lumbar spinal cords were removed for binding assays. To examine agonist-induced G-protein coupling, [35S]GTP gamma S binding experiments were performed in spinal cord membranes and sections using norepinephrine as an alpha2-adrenergic agonist. SNL was associated with an increase in maximal efficacy, but not potency, of norepinephrine-stimulated [35S]GTP gamma S binding in dorsal horn. SNL had no effect on basal [35S]GTP gamma S binding or on muscarinic cholinergic-stimulated [35S]GTP gamma S binding. [35S]GTP gamma S autoradiography showed that this increase in alpha2-adrenergic-activated G-proteins occurred both ipsilateral and contralateral to SNL surgery. SNL did not alter total alpha2-adrenergic receptor number or affinity to [3H]-rauwolscine binding, and displacement studies with the alpha2A-adrenergic antagonist BRL44408 revealed that most of the binding was associated with the alpha2A-adrenergic subtype. These data suggest that the increased potency of clonidine in neuropathic pain could reflect increased efficiency of G-protein coupling from spinal alpha2-adrenergic receptors.     

Differential inhibition by ferrous ions of [3H]MK-801 binding to native N-methyl-D-aspartate channel in neonatal and adult rat brains

In vitro addition or pretreatment with >/=1 microM ferrous chloride markedly inhibited in a concentration-dependent manner [3H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [3H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [3H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.     

Binding of [3H]prazosin and [3H]p-aminoclonidine to alpha-adrenoceptors in rat spinal cord

alpha-Adrenoceptors in spinal cord appear to play a role in a number of physiologic processes including the control of blood pressure, pain and motor function. In order to evaluate more clearly these potential roles, the characteristics of binding of [3H]prazosin ([3H]PRZ) to spinal alpha 1 adrenoceptors and [3H]p-aminoclonidine ([3H]PAC) to spinal alpha 2 adrenoceptors were determined. Binding of each ligand to their respective adrenoceptors was saturable and Scatchard analysis revealed binding of each to a single class of adrenoceptors with characteristics of [3H]PRZ binding of Bmax = 78 fmol/mg protein and Kd = 0.75 nM and [3H]PAC binding Bmax = 70 fmol/mg protein and Kd = 1.39 nM. Whereas [3H]PRZ specific binding (Bmax) was unaltered by guanine nucleotides. [3H]PAC binding was increased with addition of 10 microM guanosine triphosphate (GTP) (P less than 0.05) and decreased with either 50 microM GTP or guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] (P less than 0.01). Competition for specific [3H]PRZ and [3H]PAC binding by various alpha 1 and alpha 2 adrenoceptor agonists and antagonists of known pharmacologic activity revealed that [3H]PRZ defines alpha 1 adrenoceptors (Ki = 2.1 nM for prazosin vs 4300 nM for yohimbine) and [3H]PAC defines alpha 2 adrenoceptors (Ki = 1.06 nM for yohimbine vs 15480 nM for prazosin). Regional spinal cord studies demonstrated that dorsal spinal cord in the lumbar region contains the highest density of both [3H]PRZ (Bmax = 93 +/- 14 fmol/mg protein) and [3H]PAC (Bmax = 101 +/- 6 fmol/mg protein) binding. In contrast, lowest binding was evident in thoracic cord with equal levels in both dorsal and ventral regions (Bmax = 44-48 fmol/mg protein). The regional distribution of both alpha 1 and alpha 2 adrenoceptors in spinal cord compares to the localization previously classified functionally utilizing various pharmacological agonists and antagonists at norepinephrine receptors.     

3H]AMPA binding to glutamate receptor subpopulations in rat brain

The glutamate analog (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of L-[3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive L-[3H]glutamate binding was additive with that displaced by kainic acid (1 microM) plus N-methyl-D-aspartate (10 microM) when low concentrations of non-radioactive AMPA (1 microM) were employed to determine non-specific background, but partially overlapped when higher concentration of AMPA (100 microM) were used. [3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0-4 degrees C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of L-[3H]glutamate, and [3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with Kd values of 28 and 500 nM and Bmax values of 200 and 1800 fmol/mg protein, respectively. [3H]AMPA was inhibited by quisqualate (IC50 = 60 nM), L-glutamate (2 microM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridine-7-carboxylic acid (7-HPCA, 5 microM), kainic acid (20 microM) and glutamic acid diethyl ester (21 microM) but insensitive to L-aspartate, ibotenic acid, N-methyl-D-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 microM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [3H]AMPA binding had a similar distribution to that of L-[3H]glutamate binding displaced by AMPA (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Acetylcholine stimulates GTP binding to G proteins in rat striatum.

To evaluate the role of G proteins in acetylcholine (ACh)-mediated signal transduction, a novel assay to measure [35S]GTP gamma S binding to rat striatal membranes was standardized. ACh and carbamylcholine (CCh) stimulated the high affinity [35S]GTP gamma S binding to striatal membranes by up to 45% in a concentration-dependent manner. A time course study revealed that 0.1 microM CCh stimulated [35S]GTP gamma S binding by 26% at 4 min and 20% at 15 min. CCh-stimulated [35S]GTP gamma S binding was inhibited by atropine, N-methylscopolamine and pirenzepine. Oxotremorine (1 microM), a partial muscarinic agonist, maximally stimulated the binding to striatal membranes by only 25%.     

Alpha 1 and alpha 2 Adrenergic receptors in mouse brain astrocytes from primary cultures

Mouse brain astrocytes from primary cultures were found to contain both alpha 1 and alpha 2 adrenergic receptors. 3H WB 4101 labeled one category of binding site (KD = 1.5 +/- 0.39 nM, Bmax = 64 +/- 7.9 fmoles/mg protein) with typical alpha 1 adrenergic specificity (WB 4101 greater than prazosin greater than yohimbine). The density of alpha 1 adrenergic receptors was 2-3 times higher in mouse cerebral cortex than in glial cells. Like rat brain [U'Pritchard et al, 1979; Rouot et al, 1980], mouse glial cells were found to contain two categories of 3H clonidine binding sites: high affinity sites, which were identical to the high but not to the low affinity sites found in rat brain, since 1) they displayed the same affinity for 3H clonidine (KD = 1.2 +/- 0.13 nM, n = 4) and the same typical alpha 2 adrenergic specificity (yohimbine greater than WB 4101 greater than prazosin); 2) the dissociation rate constant for clonidine binding was equal to 0.06 min-1, a value close to that found previously for the high affinity 3H clonidine binding sites in rat brain (0.05 min-1); and 3) divalent cations augmented and guanyl nucleotides reduced 3H clonidine binding as in rat brain. Na+ decreased 3H clonidine binding in a complex manner. The number of high affinity sites in glial cells (52 +/- 9.4 fmoles/mg protein, n = 4) was half the number found in mouse cerebral cortex (98 fmoles/mg protein). Low affinity 3H clonidine binding sites (KD = 81 +/- 18 nM, Bmax = 96 +/- 5.8 fmoles/mg protein, n = 3) were not fully characterized. In conclusion, glial cells contained the same alpha adrenergic receptors as those described in brain, but their physiological function is not yet known.     

Temperature-dependent and -independent apparent binding activities of [3H]glutathione in brain synaptic membranes

An apparent binding activity of [3H]glutathione was examined by using synaptic membrane preparations of the rat brain. The activity was found to be more than two times as high at 30 degrees C as that found at 2 degrees C. At 2 degrees C, the apparent binding sites consisted of a single component with a Kd of 0.77 microM and a Bmax of 5.60 pmol/mg protein. In contrast, two independent separate sites with Kds of 0.56 and 12.6 microM and Bmaxs of 2.50 and 28.5 pmol/mg protein were observed at 30 degrees C. In vitro addition of Triton X-100 significantly inhibited the apparent binding activities detected at both temperatures, whereas pretreatment of the membranes with the detergent did not significantly affect both binding activities. Among 3 constituent amino acids of glutathione, L-cysteine induced a selective and irreversible potentiation of the apparent activities, which occurred independently of the incubation temperature. Scatchard analysis revealed that L-cysteine drastically increased the number of the low affinity sites without significantly altering their affinity. Apparent binding activities determined at both incubation temperatures were unevenly distributed in the central and peripheral structures. Distribution profile of the temperature-dependent activities was found to be closely related to that of the basal binding activity of [3H]L-glutamic acid, a putative central excitatory neurotransmitter. These results suggest that brain synaptic membranes may indeed contain specific binding sites of [3H]glutathione which have an interaction with the glutamate binding sites. Possible presence of two distinctly different apparent binding sites of [3H]glutathione, such as temperature-independent high affinity sites and temperature-dependent low affinity sites, is also suggested.