Loss of rat ventricular alpha 1-adrenergic receptors during aging

Ventricular alpha 1-adrenergic receptor concentration, measured by specific binding of [3H]-prazosin, decreases by 33% as rats age from 3 to 24 months. No age changes occur in binding affinity for [3H]-prazosin or potency of various alpha-adrenergic agonists and antagonists to displace [3H]-prazosin. The ratio of membrane protein to ventricular wet weight also does not change significantly with age. These results suggest a possible mechanism for loss of cardiovascular alpha-adrenergic responsiveness during aging.     

Beta-adrenergic binding is increased by melatonin and alpha-adrenergic compounds

Binding of the beta-adrenergic ligands [3H]dihydroalprenolol and [125I]cyanopindolol to pineal particulate fractions was increased 1- to 3.5-fold by addition of low concentrations of melatonin, alpha-adrenergic agonists, or alpha-adrenergic antagonists. Minimum concentrations of melatonin or alpha-adrenergic compounds which increased beta-adrenergic binding were between 1 pM and 0.1 nM. The increased binding of [3H]dihydroalprenolol caused by melatonin (0.1 muM) was attributed to a major increase in Bmax, which persisted in protein fractions after removal of melatonin. Melatonin enhancement of [3H]dihydroalprenolol binding was apparent after 5 to 7 min (30(0], was was optimal between 20 and 40 min, and decreased at longer times. Alpha-Adrenergic receptors are unchanged during beta-receptor enhancement.     

Synergistic Action of Postsynaptic α-Adrenergic Receptor Stimulation on Vasoactive Intestinal Polypeptide-Induced Increases in Pineal N-Acetyltransferase Activity

The alpha-adrenergic agonists phenylephrine and methoxamine, at concentrations that have little effect on pineal N-acetyltransferase activity, markedly enhance stimulation of this enzyme by vasoactive intestinal polypeptide (VIP). This augmentation can be blocked by the alpha 1-adrenergic antagonists phenoxybenzamine and prazosin and, at 10 but not 1 microM, by the alpha 2-antagonist yohimbine. The time course for VIP stimulation is not altered by concomitant alpha-adrenergic stimulation. Augmented activity does not require concomitant alpha-adrenergic stimulation, but alpha-adrenergic agonists must be present for augmentation to be maintained. Phorbol 12,13-diacetate or -dibutyrate but not 4 alpha-phorbol can substitute for phenylephrine, a finding suggesting that protein kinase C is involved in the augmentation. These results are, in general, analogous to alpha-adrenergic magnification of N-acetyltransferase induction by beta-adrenergic agonists.     

Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors.     

Inhibition of the lipolytic action of beta-adrenergic agonists in human adipocytes by alpha-adrenergic agonists

The aim of this study was to define the role of the alpha-adrenergic receptor in the regulation of lipolysis by human adipocytes. Glycerol production by isolated human adipocytes was stimulated by the pure beta-adrenergic agonist isoproterenol in a dose-dependent fashion. This stimulation of lipolysis was inhibited by the alpha-adrenergic agonists methoxamine, phenylephrine, and clonidine. Epinephrine-stimulated lipolysis was potentiated by the alpha-adrenergic antagonists, dihydroergocryptine, phentolamine, phenoxybenzamine, and yohimbine. Whereas the attenuation of beta-adrenergic agonist-stimulated lipolysis by alpha-adrenergic agonists was reversed completely by the alpha 2-adrenergic antagonist yohimbine, the alpha 1-antagonist prazosin did not reverse such attenuation. It is concluded that alpha-adrenergic agonists act as antilipolytic agents in human adipocytes and that this action may result from the interaction of these compounds with a population of alpha 2-adrenergic receptors.     

Amitriptyline binding to specific alpha-adrenergic receptors in vitro

Amitriptyline was found to compete with (³H) dihydroergocryptine (³H-DHE), a potent alpha adrenergic antagonist, for specific alpha-adrenergic binding sites in rabbit uterine membranes preparation. Amitriptyline and adrenergic agonists compete for (³H) DHE binding sites in the following order of potency : (?) epinephrine > (?) nor-epinephrine > amitriptyline > ethylphenylephrine. Amitriptyline and alpha and beta antagonists compete in the order : phentolamine ? phenoxybenzamine > amitriptyline > propranolol. Based on 50 per cent inhibition values from binding competition curves, the affinity of amitriptyline for alpha-adrenergic sites was 83-fold lower than phentolamine, but only ten fold lower than epinephrine. These data are consistent with the hypothesis that the hypotensive action of amitriptyline results at least in part from alpha-adrenergic blockade.     

Postsynaptic localization of the alpha receptor-mediated stimulation of phosphatidylinositol turnover in pineal gland.

The phosphatidylinositol effect in rat pineal gland (defined as the enhanced incorporation of P_i into phosphatidylinositol which is elicited by agonists) is mediated through alpha-adrenergic receptors. Experiments with glands from animals treated with 6-hydroxydopamine, with dispersed pinealocytes and with a number of relatively specific pre- and postsynaptic alpha-adrenergic agonists and antagonists have shown the receptors involved to be located at postsynaptic sites.     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

Alterations in cardiovascular responsiveness and adrenoceptor binding during catecholamine infusion hypertension in rats.

Chronic continuous infusion of norepinephrine in rats causes alterations in biochemical and physiologic responses of the cardiovascular system and in cardiovascular adrenoceptor number. The response of cardiac and aortic ornithine decarboxylase (ODC) activity to stimulation by norepinephrine was decreased in rats receiving norepinephrine infusion. These responses are due to stimulation of beta- and alpha-adrenergic receptors, respectively. Additionally, there was reduced stimulation of aortic ODC activity by angiotensin II and vasopressin. The cardiac ODC response to angiotensin II was decreased, but the response to vasopressin was not affected. The decreased ODC response is accompanied by decreased pressor responses to the alpha-adrenergic agonist phenylephrine. Decreased numbers of alpha- and beta-adrenoceptor binding sites (as measured by the binding of [3H]prazosin and [125I]pindolol) might mediate, in part, the altered responses to adrenergic agonists. The decreased cardiovascular responsiveness measured in these animals after several days of norepinephrine infusion hypertension contrasts with the increased responses found in most other forms of hypertension. This provides a useful model in which to examine the consequences of prolonged adrenergic receptor stimulation.     

In vitro studies on the effects on granular gland secretion in Xenopus laevis skin of stimulation and blockade of alpha and beta adrenoceptors of myoepithelial cells.

Secretion from the granular glands of Xenopus laevis skin was stimulated by alpha-adrenergic agonists, an effect which was blocked by alpha-adrenergic antagonists and inhibited by beta-adrenergic agonists, db-cAMP and diazoxide. The inhibition by isoprenaline and salbutamol, but not that by diazoxide, was blocked by a beta-adrenergic antagonist. It is concluded that the myoepithelial cells surrounding the secretory compartment of the granular glands bear alpha and beta adrenoceptors, and that the beta receptors comprise, or at least include beta2 receptors.     

Alpah-adrenergic receptor modulation of beta-adrenergic, adenosine and prostaglandin E1 increased adenosine 3':5'-cyclic monophosphate levels in primary cultures of glia.

Beta-adrenergic agonists, adenosine and prostaglandin E1 increased the level of adenosine 3':5'-monophosphate (cAMP) in glial cultures prepared from rat cerebral cortical tissue. In addition to these physiological effectors, cholera toxin also increased cAMP levels in these cultures. The accumulation of cAMP in response to each of these agen-s, including cholera toxin, was partially blocked (50--80%) by simultaneous alpha-adrenergic receptor stimulation. Basal levels of cAMP were not affected by alpha-adrenergic agonists. These results indicate that in glia, alpha-adrenergic receptors may serve to modulate the level of cAMP which normally accumulates in response to a number of neurohumoral substances. The modulatory effect of alpha-adrenergic agents does not appear to reduce cAMP accumulation by activating phosphodiesterase since the effect was not blocked by a potent inhibitor of this enzymemthe results suggest that the modulatory effect of alpha-adrenergic receptor activation results from an interaction which takes place at some point in between adenylate cyclase-associated-membrane receptors and the enzymatic degradation of cAMP.     

Absence of alpha-adrenergic inhibition of lipolysis in swine adipose tissue

Adipose tissue slices from young and older pigs and genetically obese pigs were incubated to demonstrate alpha-adrenergic inhibition of lipolysis as found by other investigators in dog, guinea-pig, hamster, human and rabbit adipose tissue. Purported alpha-adrenergic agonists (amidephrine, clonidine, methoxamine, phenylephrine) did not inhibit basal or catecholamine-stimulated lipolysis. Purported alpha-adrenergic antagonists (dihydroergotamine, phenoxybenzamine, phentolamine, prazosin, yohimbine) did not enhance basal or stimulated lipolysis. Adipose tissue from pigs is different from that of most species but similar to that of rats with no alpha-adrenergic inhibition of lipolysis.     

Alpha-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes. Similarity of receptor with alpha-2 adrenergic receptors

The present communication shows the effects of several alpha-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 micrograms/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the alpha-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 microM methoxamine; 10 microM methyl norepinephrine; 10 microM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of alpha-adrenergic blocking drugs on prevention of the inhibitor effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the alpha-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to alpha-2 adrenergic receptors.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Characterization by [3H]Dihydroergocryptine Binding of Alpha-Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.     

Comparative effect of atropine on the adrenergic and muscarinic stimulation of phospholipid 32P labelling in isolated parotid cells: atropine, a possible blocker of alpha-adrenergic receptors

The inhibitory effect of atropine on phospholipid 32P labelling stimulated by muscarinic or alpha-adrenergic agonists was studied in isolated parotid cells. Atropine (10(-11) to 10(-4) M) had no effect on phospholipid 32P labelling in unstimulated cells. In contrast, 10(-8) to 10(-7) M atropine provoked a competitive inhibition of the cholinergic stimulation (i.e. this effect was completely wiped out at high agonist concentration). The atropine app. KD for the muscarinic receptor was 5 X 10(-9) M. Moreover, atropine inhibits the adrenergic stimulation of phospholipid 32P labelling by decreasing the efficacity and potency of the adrenergic agonists. The atropine app. KD for the alpha-adrenergic receptor can be estimated at 10(-5) M. This inhibition of alpha-adrenergic stimulation appears to be specific since atropine was without effect on the substance P or beta-adrenergic stimulation. At very low concentration (10(-10) - 10(-9) M) atropine seems to be a modulator (activator) of the muscarinic or adrenergic agonist-receptor complex. From the present data, it is suggested that atropine, besides its classical blocker effect at the muscarinic receptor, at high concentration is a specific alpha-adrenergic antagonist.     

Norepinephrine-induced down regulation of alpha 1 adrenergic receptors in cultured rabbit aorta smooth muscle cells

Drug-induced refractoriness of alpha-adrenergic receptor-mediated vasoconstriction may be a clinically important phenomenon. We have investigated the possible molecular mechanisms underlying this phenomenon in cultured vascular smooth muscle cells derived from the rabbit aorta. alpha 1-Adrenergic receptors were identified in membranes prepared from these cells by [125I]HEAT binding. The radioligand bound to a high affinity site (Kd = 140 pM) in a saturable fashion (202 fmol/mg protein). Adrenergic agonists and antagonists competed for binding of [125I]HEAT with the expected order of potency for an alpha 1-receptor, (-)epinephrine greater than or equal to (-) norepinephrine greater than (+)epinephrine greater than isoproterenol and prazosin greater than phentolamine greater than yohimbine. Exposure of cells for 26 hours to 10 microM norepinephrine resulted in a 70% decrease in the number of alpha 1-receptors as measured by [125I]HEAT binding without any significant change in the affinity of the receptor for the ligand. When the alpha-receptors were blocked with 10 microM phentolamine the loss of receptors induced by norepinephrine was completely prevented. Similar down-regulation of the [125I]HEAT binding sites was observed when the alpha 1-agonist phenylephrine was used instead of norepinephrine. It is concluded that alpha-agonists induce down-regulation of aortic smooth muscle alpha 1-receptors. This reduction of alpha-receptors could be important in the mechanisms by which vascular smooth muscle develops refractoriness to alpha-adrenergic stimulation.     

The action of alpha-adrenergic agonists on plasma-membrane calcium fluxes in perfused rat liver

The effect of alpha-adrenergic agonists on Ca2+ fluxes was examined in the perfused rat liver by using a combination of Ca2+-electrode and 45Ca2+-uptake techniques. We showed that net Ca2+ fluxes can be described by the activities of separate Ca2+-uptake and Ca2+-efflux components, and that alpha-adrenergic agonists modulate the activity of both components in a time-dependent manner. Under resting conditions, Ca2+-uptake and -efflux activities are balanced, resulting in Ca2+ cycling across the plasma membrane. The alpha-adrenergic agonists vasopressin and angiotensin, but not glucagon, stimulate the rate of both Ca2+ efflux and Ca2+ uptake. During the first 2-3 min of alpha-agonist administration the effect on the efflux component is the greater, the net effect being efflux of Ca2+ from the cell. After 3-4 min of phenylephrine treatment, net Ca2+ movements are essentially complete, however, the rate of Ca2+ cycling is significantly increased. After removal of the alpha-agonist a large stimulation of the rate of Ca2+ uptake leads to the net accumulation of Ca2+ by the cell. The potential role of these Ca2+ flux changes in the expression of alpha-adrenergic-agonist-mediated effects is discussed.     

Effects of epinephrine, clonidine, L-phenylephrine, and morphine on intestinal secretion mediated by Escherichia coli heat-stable enterotoxin in pig jejunum

Perfusion of pig jejunum with Escherichia coli heat-stable enterotoxin (strain 1261) reversed net absorption of water and electrolytes to net secretion. Addition of the alpha-adrenergic agonists clonidine (5 X 10(-7) M) or L-phenylephrine (5 X 10(-6) M), or the opiate agonist morphine (3.6 X 10(-6) M) to the perfusate reduced the secretory response to enterotoxin and stimulated absorption in normal jejunum. Epinephrine (5 X 10(-5) M) did not stimulate absorption in controls but reduced chloride loss in the presence of enterotoxin. Mucosal sodium--potassium adenosine triphosphatase was unchanged but disaccharidase activity was decreased in the presence of enterotoxin. The results suggest that alpha-adrenergic agonists and opiate agonists may exert an antidiarrheal action by increasing net transport across intestinal epithelium.     

Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin

Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4026-4030). The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex. These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.