Differential effects of neonatal norepinephrine lesions on immediate early gene expression in developing and adult rat brain.

Activity regulated cytoskeletal protein (Arc), c-fos and zif268 are immediate early genes (IEGs) important for adult brain plasticity. We examined developmental expression of these IEGs and the effect of neonatal noradrenergic lesion on their expression in developing and mature brain. N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), a specific noradrenergic neurotoxin, was administered to rats on postnatal day (PND) 3 and in situ hybridization was used to assay Arc, c-fos and zif268 mRNA on PND 13, 25 and 60. In contrast to decreases in Arc, c-fos and zif268 expression produced by noradrenergic lesions of mature brain, lesions on PND 3 yield a strikingly different effect. Neonatal lesions produce increases in c-fos and zif268 expression in specific frontal cortical layers on PND 13, while Arc shows no change. These lesions lead to increases in zif268 expression in frontal cortical layers on PND 25, with no changes in c-fos or Arc expression, and on PND 60 they produce a significant increase in c-fos expression in hippocampus with no significant changes in Arc or zif268 expression. 2-[2-(2-Methoxy-1,4-benzodioxanyl)]imidazoline hydrochloride (RX821002), an alpha-2 adrenergic receptor (A2AR) antagonist, administered to control PND 60 animals produces elevations of Arc, zif268 and c-fos mRNAs. This response was eliminated in animals lesioned with DSP-4 on PND 3. These data indicate that norepinephrine regulation of IEG expression differs in developing and mature brain and that loss of developmental norepinephrine leads to abnormally high postnatal IEG expression. Previous studies have shown an important role for norepinephrine in brain development. Our data support the idea that norepinephrine plays an important role during CNS development and that changes in noradrenergic signaling during development may have long lasting effects, potentially on learning and memory.

Pharmacological characterizations of adrenergic receptors in human adipocytes.

Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [(3)H]dihydroalprenolol binding (beta adrenergic) B(max) 280 fmol/mg protein, K(D) 0.38 nM; [(3)H]para-aminoclonidine binding (alpha-2 adrenergic) B(max) 166 fmol/mg protein, K(D) 0.49 nM; [(3)H]WB 4101 binding (alpha-1 adrenergic) B(max) 303 fmol/mg protein, K(D) 0.86 nM. In adipocytes from subcutaneous adipose tissue, [(3)H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 muM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 muM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [(32)P]phosphate and epinephrine (10 muM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in (32)P incorporation into phosphatidylinositol. Prazosin (0.1 muM) blocked this action whereas yohimbine (0.1 muM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.

Development of the norepinephrine transporter in the rat CNS.

The norepinephrine transporter (NET) plays a major role in regulating the actions of norepinephrine by removing norepinephrine from the synapse. Many studies suggest norepinephrine plays an important role in regulating development of the CNS, pointing to NET as an important factor in this process. We examined the ontogeny of NET expression in rat brain at 5, 10, 15, 20 and 25 days postnatally (PND) and in adults, using quantitative autoradiography with [3H]nisoxetine as ligand. At PND 5 and 10 most forebrain areas had low NET expression (1-2 fmol/mg tissue). By PND 15 most forebrain areas increased NET expression approximately five-fold compared with PND 10, levels generally similar to those found in the adult brain. In contrast, NET development in the brainstem exhibited elevated densities at PND 5, 10 and 20 that decreased in the adult. The locus coeruleus, in particular, had very high NET expression in the early postnatal period that decreased dramatically in the adult brain. These data illustrate a dynamic ontogenic profile for NET, characterized by developmental increases in forebrain structures and contrasting decreases in the brainstem. The early postnatal expression of NET in brainstem and the subsequent decrease or loss of NET expression with maturation suggest an important role for this transporter and for norepinephrine in the development of many brain regions. These studies also have important implications for use of drugs targeting the noradrenergic system in children and adolescents, such as antidepressants and drugs of abuse.

Neurotransmitter receptors in frontal cortex of schizophrenics.

Frontal cerebral cortex brain samples from schizophrenics and controls have been assayed for binding associated with muscarinic cholinergic, serotonin (5HT), gamma-aminobutyric acid (GABA), and beta-adrenergic receptors as well as for the activity of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Binding levels of tritium-LSD, presumably associated with postsynaptic 5HT receptors, were reduced 40% to 50% in samples from schizophrenics in three independent studies, whereas no other consistent alteration was observed in levels of binding associated with other receptors or in the activity of GAD. This change in receptor binding levels does not seem to be attributable to postmortem changes, to influence of drugs received by the patients, or to demographic features of the patient populations.

Urinary MHPG and HVA excretion in boys with attention deficit disorder and hyperactivity treated with d-amphetamine.

The authors examined the excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG) and homovanillic acid (HVA) in nine hyperactive and nine control boys admitted to a clinical research center. The hyperactives excreted lower 24 hr MHPG and HVA levels than controls. d-Amphetamine 0.5 mg/kg body weight daily for 2 weeks decreased MHPG and increased HVA. These data replicate the authors' previous findings on MHPG and HVA and on the effect of d-amphetamine in hyperactive children. The data suggest the involvement of norepinephrine and dopamine in the etiology of hyperactivity. It further suggests d-amphetamine may achieve its clinical effects in hyperactivity by inhibiting NE and potentiating DA activity.

alpha 2-Adrenergic receptor binding in human platelets: alterations during the menstrual cycle.

We studied the effect of several clinically important variables on the characteristics of alpha 2-adrenergic receptors in human platelet membranes. The number and affinity of the receptor binding sites were determined from radioligand binding experiments, with [3H]yohimbine being the radioligand of choice. Platelets from female subjects had a cyclic variation in the number of alpha 2-adrenergic receptors that coincided with their menstrual cycles. The number of alpha 2-receptors was highest at the onset of menses and dropped to 74% to 79% of that value during the middle of the cycle. In concurrent experiments we did not observe comparable cyclic changes in receptor binding sites in platelets from male subjects. There was no age-dependent alteration in receptor number in a sample of 39 subjects ranging in age from 8 to 80 yr, but the number of alpha 2-receptors in platelets from male and female subjects differed. We also tested the possibility of a circadian rhythm in alpha 2-receptor number but found no cyclic changes as a function of time of day. There was no alteration in alpha 2-adrenergic receptor binding in the platelets from five subjects with Parkinson's disease. Finally, there was no change in receptor affinity as a function of any of the variables tested. These data should apply to the design of further studies on the clinical importance of platelet alpha 2-adrenergic receptors.

Platelet MAO in children with attention deficit disorder and hyperactivity: a pilot study.

The authors examined platelet MAO activity in 8 hyperactive and 18 control children who were admitted to a clinical research center and placed on a low monoamine diet. After 5 days, their blood was analyzed; the hyperactive children were discharged on day 7, placed on d-amphetamine for 2 weeks, and readmitted for repeat blood analysis. The hyperactive children initially had significantly lower levels of platelet MAO than the controls. After the hyperactive children were treated with d-amphetamine for 2 weeks, their platelet MAO levels were comparable to those of the control children. The authors suggest an association between low platelet MAO activity and a behavioral state of overactivity, short attention span, and impulsivity.

Alpha-2 adrenergic receptor development in rat CNS: an autoradiographic study.

During development norepinephrine plays a role in determining the morphologic organization of the CNS and the density and future responsiveness of adrenergic receptors. alpha-2 Adrenergic receptors, one of three adrenergic receptor types, regulate important adult CNS functions and may have a distinct role during development. We examined alpha-2 receptor distribution and density in the rat brain at postnatal days 1, 5, 10, 15, 21, 28 and in adults using the antagonist [(3)H]RX821002 for autoradiography. Binding kinetics and pharmacology for alpha-2 adrenergic receptors were the same in adults and neonates. There was an overall increase in alpha-2 receptor levels during postnatal development with great variability in pattern and timing of receptor density changes among brain regions. Three major patterns were apparent. First, in many regions receptor density increased during postnatal development, generally reaching adult levels around postnatal day 15. Within this group there was variability in timing between regions and there were several regions with receptor densities higher than adult levels during the postnatal period. Second, there were regions with very high levels of receptors at birth and little or no change in density during the postnatal period. Third, some regions demonstrated decreasing or transient expression of alpha-2 adrenergic receptors in the course of postnatal development, including white matter regions, cerebellum and many brainstem nuclei, suggesting specific roles for alpha-2 receptors during development. This study investigates the development of alpha-2 adrenergic receptors in the rat CNS. It demonstrates there is region-specific regulation of alpha-2 receptor development and identifies brain regions where these receptors may play a specific and critical role in the regulation normal development.

Characterization of alpha2 adrenergic receptor subtypes in human ocular tissue homogenates.

PURPOSE: To determine the predominant alpha2 adrenergic receptor subtypes present in the human eye. METHODS: Saturation- and competition-receptor- binding experiments were performed with the radioligand [3H]RX821002 in human ciliary body, retinal pigmented epithelium-choriocapillaris, iris, and neurosensory retina. The affinities of various adrenergic antagonists in these ocular tissues were compared with their affinities for the cloned alpha2A, alpha2B, and alpha2C adrenergic receptor subtypes. RESULTS: The density of alpha2 adrenergic receptors was highest in the iris (440 femtomoles/mg protein), lowest in the neurosensory retina (14 femtomoles/mg protein), and intermediate in the other two tissues (approximately 90 fmol/mg protein). The drug affinities in all four human ocular tissues were highly correlated (correlation coefficients between 0.94 and 0.97) with the affinities for the human alpha2A adrenergic receptor subtype and poorly correlated (correlation coefficients between 0.15 and 0.66) with the alpha2B and alpha2C subtypes. CONCLUSIONS: In agreement with previous studies in several animal species, the alpha2 adrenergic receptors in the human ciliary body, retinal pigmented epithelium-choriocapillaris, iris, and neurosensory retina are predominately of the alpha2A subtype.

Alpha-2 adrenergic receptors in the bovine retina. Presence of only the alpha-2D subtype.

PURPOSE: To identify and characterize the alpha-2 adrenergic receptor subtypes present in the bovine neurosensory retina. METHODS: Radioligand saturation and inhibition binding assays were performed with the antagonist radioligands [3H]RX821002 and [3H]rauwolscine. RESULTS: [3H]RX821002 bound to a single class of receptors with the characteristics of an alpha-2 adrenergic receptor with an affinity (KD) of 0.16 nM and a receptor density (Bmax) of 1500 fmol/mg protein. Correlation of the affinities (pKi values) for nine antagonists in the bovine neurosensory retina with the alpha-2D receptor of the bovine pineal gave a correlation coefficient of 0.99. The correlation coefficients for the alpha-2A (0.84), alpha-2B (0.36), and alpha-2C (0.39) subtypes were much lower. The presence of a minor population of alpha-2B or alpha-2C receptors was excluded. CONCLUSIONS: A high density of alpha-2D adrenergic receptors is present in the bovine neurosensory retina. Neither the alpha-2B nor the alpha-2C subtype is detectable.

Ascorbic acid inhibition of alpha-adrenergic receptor binding.

Relatively low concentrations of ascorbic acid inhibited the binding of the alpha-1 adrenergic antagonist [125I]HEAT [DL-[beta(3-iodo-4-hydroxyphenyl)-ethyl-aminomethyl]-tetralone) in rat submandibular gland and rat aorta. However, no inhibition was observed with this ligand in several other tissues, nor with several other ligands in these tissues. The inhibition observed was dependent on the concentration of both the ascorbic acid and the tissue. Maximal inhibition of [125I]HEAT occurred in submandibular gland at 10 microM ascorbic acid with Bmax values reduced 65% and no change in affinity. Ascorbic acid had a greater effect in assays in which less tissue was used, causing a 22% decrease in binding at 46 micrograms/ml, but a 48% decrease in binding at a tissue concentration of 12 micrograms/ml. EDTA prevented the loss of binding normally seen with ascorbic acid at a tissue concentration of 17 micrograms/ml. We suggest that, if an antioxidant is thought to be necessary in an assay system, its effects be carefully examined before routine use.

Effects of alpha 2-adrenergic agonist preincubation on subsequent forskolin-stimulated adenylate cyclase activity and [3H]forskolin binding in membranes from HT29 cells.

alpha 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [3H]forskolin binding experiments, alpha 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.

Binding of [3H]flunitrazepam to the LM cell, a transformed murine fibroblast.

LM cells have a saturable, high affinity binding site for [3H]flunitrazepam with a KD of 13 nM and a Bmax of 19 pmoles/mg protein. The IC50 values for Ro 5-4864, flunitrazepam and clonazepam against [3H]flunitrazepam were 6, 23 and 2800 nM, respectively, indicating that this receptor is of the peripheral type. A decrease of 37, 26 and 26% in Bmax was associated with substituting dimethylethanolamine, monomethylethanolamine or ethanolamine, respectively, for choline in the cell culture medium. These treatments did not change either the KD of [3H]flunitrazepam binding or the IC50 values of the different benzodiazepine drugs. Metastatic cell lines of the LM cell obtained from either athymic or C3H/Hef mice exhibited alterations in the binding parameters of [3H]flunitrazepam. There was a reduction in the Bmax values of the athymic (34%) and the C3H/Hef (44%) cell lines compared to the LM cell. In both groups there was a 90% increase in the KD. In the C6 astrocytoma, the peripheral type receptor appears to regulate plasma membrane mediated synthesis of phosphatidylcholine from phosphatidylethanolamine. However, this was not observed in the LM cell. Nor did it modulate cyclic AMP metabolism as assessed by measurement of cyclic AMP levels in whole cells after drug treatment.

Effects of dihydropyridine derivatives and anticonvulsant drugs on [3H]nitrendipine binding and calcium and sodium fluxes in brain.

The binding of [3H]nitrendipine to rat cortical membranes was reduced by phenytoin, phenobarbital, and pentobarbital. The IC50 values were 0.09, 0.40, and 0.76 mM respectively. The drugs reduced the apparent binding affinity of [3H]nitrendipine with little effect on the maximum number of binding sites. The inhibitory effects of the drugs were similar in the absence and presence of calcium (4.5 mM). Neither nimodipine (10(-8) to 10(-5) M) nor nifedipine (10(-8) to 10(-7) M) altered the voltage-dependent uptake of 45Ca2+ by synaptosomes from rat cortex. Phenytoin inhibited 45Ca2+ influx, and this inhibition was not altered by nifedipine. Nimodipine and nifedipine (10(-6) M) produced a small inhibition of the voltage-dependent uptake of 24Na+ by synaptosomes. Ethanol, phenytoin or pentobarbital reduced 24Na+ influx, and this action was not altered by nimodipine. Thus, sedative-anticonvulsant drugs reduced the binding of dihydropyridines to brain membranes, but these interactions did not appear to involve either calcium or sodium channels.

Pharmacological characteristics of alpha-2 adrenergic receptor subtypes.

The adrenergic receptor family has three main members: alpha-1, alpha-2, and beta receptor types. Each of these three types contains three or more subtypes. There are currently four pharmacological (2A, 2B, 2C, and 2D) and three molecular (or genetic) alpha-2 adrenergic subtypes (2A/D, 2B, and 2C). Because different functions are likely to be mediated by different subtypes, much effort is being directed towards understanding the physiological roles of the various subtypes. However, little is currently known about the specific function mediated by the various subtypes.

Studies on desensitization of adrenergic receptors of human adipocytes.

The studies described here were undertaken to determine whether or not desensitization of human adipocyte beta and alpha-2 adrenergic receptors could be demonstrated. Cells, isolated from peritoneal adipose tissue obtained from patients undergoing elective abdominal surgery, were preincubated for 3 hr in buffer alone or in the presence of isoproterenol, 10-5M. Cells in both sets of flasks were then washed and exposed to isoproterenol for 1/2 hr; cyclic AMP was then measured as an end point of beta receptor activation. Cells which had had no prior exposure to isoproterenol responded significantly greater to isoproterenol than did cells that had had prior exposure to the catecholamine, The beta receptor characteristics of cells undergoing beta desensitization were assessed using [3H] dihydroalprenolol. Compared to control cells, adipocytes exposed to isoproterenol had a reduction in Bmax while KD values were the same. Thus desensitization of beta adrenergic receptors of human adipocytes occurs and is associated with down regulation in the number of beta receptors. In comparable studies, preincubation with epinephrine 10-5M did not affect the response of cells to a subsequent exposure to this catecholamine. In alpha-2 receptor binding assays, there was a decreased number of [3H]p-aminoclonidine binding sites, but the level of [3H]yohimbine binding was not altered following the incubation with epinephrine. Thus, desensitization of alpha-2 receptors was not demonstrated.

A fragment of vasoactive intestinal peptide, VIP(10-28), is an antagonist of VIP in the colon carcinoma cell line, HT29.

The 19 amino acid carboxyl terminus fragment of vasoactive intestinal peptide (VIP), VIP(10-28), inhibits [125I]VIP binding in intact HT29 colonic adenocarcinoma cells and in membranes from these cells. However, VIP(10-28) alone has no effect on adenylate cyclase activity (membranes) or cyclic AMP synthesis (intact cells) in HT29 cells although VIP receptor agonists are markedly stimulatory. The indicated antagonist character of VIP(10-28) was confirmed by rightward parallel shifts of VIP dose response curves in the presence of VIP(10-28) in adenylate cyclase and cyclic AMP synthesis experiments. The equilibrium dissociation constant values for VIP(10-28) from these experiments agree with values from inhibition binding studies. The lack of effect of VIP(10-28) on forskolin dose response curves in HT29 adenylate cyclase assays indicates the specificity of the VIP(10-28) antagonism, thus suggesting that VIP(10-28) may be a useful tool in studying VIP receptor regulation and other aspects of the mechanisms of VIP action. The potential regulatory role of a proteolytically generated fragment of VIP acting antagonistically at VIP receptors is discussed.

A robust GTP-induced shift in alpha(2)-adrenoceptor agonist affinity in tissue sections from rat brain.

A method is presented for monitoring the coupling of the alpha(2)-adrenoceptor, as well as other receptors, to their G proteins using the GTP-induced shift in agonist affinity states. In tissue sections GTP, but not ATP, induces a robust decrease in agonist affinity of greater than 100-fold, which is much larger than previously found in membrane preparations. A sensitive and easy procedure to monitor the extent of coupling is to compare the amount of [(3)H]RX821002 binding remaining in the presence of 100 nM brimonidine in the absence and presence of 100 microM GTP. This method should be especially applicable for determining the extent of coupling of receptors to their G proteins in multiple brain regions using autoradiographic procedures.

Agonist-stimulated [35S]GTPgammaS autoradiography: optimization for high sensitivity.

The receptor-stimulated accumulation of [35S]GTPgammaS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [35S]GTPgammaS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [35S]GTPgammaS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for mu-opiate receptors, 5-HT(1A) receptors and alpha(2)-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [35S]GTPgammaS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [35S]GTPgammaS incubation time (2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and use of [35S]GTPgammaS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [35S]GTPgammaS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [35S]GTPgammaS autoradiography to receptors, which produce low levels of [35S]GTPgammaS binding and to the measurement of changes in receptor-G protein coupling.