Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

Changes in the response to adrenergic drugs on mouse uterine contractions during pregnancy

The effect of isoproterenol (ISO), norepinephrine (NE) and phenylephrine (PHE) on electrically-induced contractions of mice uterine horns was studied during pregnancy. At the different times of gestation adrenergic agonists always inhibited uterine contractions in the following rank order of potency: ISO greater than NE greater than PHE. Cumulative dose-response curves constructed for the effect of these amines during diestrous, and at days 3-7, 10-15, 17-21 of gestation, showed that EC50 values increased gradually as term approached, which could imply a lower capacity of the uterus to respond to adrenergic drugs. Some likely explanations for this phenomenon are proposed. It is suggested that this lower response to catecholamines at the end of pregnancy could be a cause for the reduced success of beta 2-adrenergic drugs to stop premature labor.     

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.     

Subtypes of beta adrenergic receptors mediating pigment dispersion in chromatophores of the medaka, Oryzias latipes

Actions of the adrenergic beta-2 agonists, salbutamol and terbutaline, and the beta-1 antagonists, metoprolol and atenolol, were examined on denervated melanophores and leucophores of a teleost, Oryzias latipes. Beta-2 agonists depressed the pigment-aggregation response of melanophores to norepinephrine, while beta-1 antagonists inhibited the dispersion response of leucophores to isoproterenol but not the melanophore response. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.     

Multiple Adrenergic Receptor Subtypes Controlling Cyclic AMP Formation: Comparison of Brain Slices and Primary Neuronal and Glial Cultures

The adrenergic receptor subtypes involved in cyclic AMP responses to norepinephrine (NE) were compared between slices of rat cerebral cortex and primary neuronal and glial cultures from rat brain. In neuronal cultures, NE and the beta-adrenergic receptor agonist isoproterenol (ISO) caused similar increases in cyclic AMP, which were not altered by the alpha-adrenergic receptor antagonist phentolamine. In glial cultures, NE caused a much smaller cyclic AMP response than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists (phentolamine greater than yohimbine greater than prazosin). alpha 2-Adrenergic receptor agonists partially inhibited the ISO response in glial cultures to a level similar to that observed with NE alone (clonidine = UK 14,304 greater than NE greater than 6-fluoro-NE greater than epinephrine). In slices from cerebral cortex, NE caused a much larger increase in cyclic AMP than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists with a different order of potency (prazosin greater than phentolamine greater than yohimbine). alpha 1-Adrenergic receptor agonists potentiated the response to ISO to a level similar to that observed with NE alone (epinephrine = NE greater than phenylephrine greater than 6-fluoro-NE greater than methoxamine). In all three tissue preparations, large responses to both alpha 1-receptor activation (increases in inositol phosphate accumulation) and alpha 2-receptor activation (decreases in forskolin-stimulated cyclic AMP accumulation) were observed. These data indicate that all of the major adrenergic receptor subtypes (beta, alpha 1, alpha 2) are present in each tissue preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated enhancement of beta adrenergic drug activity by ascorbate in vitro and in vivo

Rationale

Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors.

Objectives

Extending this work to beta 2 adrenergic systems in vitro and in vivo.

Methods

Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction.

Measurements

Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep.

Main Results

Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3–10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol''s effect on heaves and produces a 10-fold enhancement of albuterol activity in “asthmatic” sheep.

Conclusions

Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications.     

Pharmacological evidence for adrenergic activation of hyperthermic responses associated with psychosocial stimulation

1. Brief social encounters between strange voles living adjacent compartments of a population cage lead to rapid increases of more than 1 degrees C in their core temperatures. 2. Socially induced core temperature responses remain elevated for more than 1 hr following social contact, and are of greater amplitude and duration than can be elicited by handling, noise or exercise. 3. Pretreatment of opponents with adrenergic blockers reduces the amplitude and duration of hyperthermic response proportional to drug intake; both alpha and beta blocking effects are completely reversed within 24 hr. 4. The dynamics of the hyperthermic response and effects of the drugs suggest that the rapid rise in core temperature is due to both vasoconstrictor (alpha) heat conservation and metabolic (beta) heat generating mechanisms, and that the protracted elevation of core temperature is primarily due to (beta) metabolic activation.     

The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.     

Adrenergic control of lipogenesis and lipolysis in the chicken in vitro

The adrenergic inhibition of lipogenesis and stimulation of lipolysis in the avian has been examined using chicken hepatocytes and adipose tissue explants in vitro. Lipogenesis was inhibited by adrenergic agonists: epinephrine (alpha + beta) greater than isoproterenol (beta 1/beta 2) greater than norepinephrine (alpha 1/alpha 2, beta 1) greater than metaproterenol (beta 2), phenylephrine (alpha 1). Dobutamine (beta 1 agonist) and dopamine (dopaminergic agonist) did not significantly affect [14C]acetate incorporation into lipid, while clonidine and para-aminoclonidine (alpha 2 agonists) were slightly stimulatory. Lipolysis in young and adult chicken adipose tissue was stimulated by epinephrine, isoproterenol, phenylephrine, dobutamine and metaproterenol, but was inhibited by clonidine and para-aminoclonidine. Both the antilipogenic and lipolytic effects of epinephrine were partially blocked by phentolamine (alpha 1 = alpha 2 antagonist) or propranolol (beta 1 = beta 2 antagonist), but completely inhibited by phentolamine and propranolol administered together.     

Apparent absence of alpha-2 adrenergic receptors from hamster brown adipocytes

The possible presence of α adrenergic control of lipolysis and cyclic AMP production in brown adipocytes of hamsters was studied in adipocytes isolated from interscapular, subscapular, cervical and axillary regions of normal male hamsters maintained at 25°C. Lipolysis activated by either 3-isobutyl-1-methyl xanthine or isoproterenol was unaffected by the presence of the α adrenergic selective agonists clonidine and methoxamine. Similarly, accumulation of cyclic AMP in response to β-receptor stimulation, alone or in combination with a methyl xanthine, was unaffected by clonidine or methoxamine. In contrast, both lipolysis and cyclic AMP accumulation in brown fat cells were effectively suppressed in the presence of nicotinic acid, prostaglandin E₁ or N⁶-phenylisopropyl adenosine. Accumulation of cyclic AMP in response to the mixed agonist norepinephrine was not influenced when cells were exposed to the alpha adrenergic blocking drugs yohimbine or tolazoline. These observations suggest that alpha-2 adrenergic receptors which are present on hamster white fat cells and control production of cyclic AMP and lipolysis are absent from hamster brown adipocytes. On the other hand, brown fat cells of this species appear to respond to a number of other inhibitory compounds in a manner not markedly different from that of white adipocytes.     

Sex-dependent thermogenesis,differences in mitochondrial morphology and function,and adrenergic response in brown adipose tissue

Gender-related differences in brown adipose tissue (BAT) thermogenesis of 110-day-old rats were studied by determining the morphological and functional features of BAT. The adrenergic control was assessed by studying the levels of beta(3)- and alpha(2A)-adrenergic receptors (AR) and by determining the lipolytic response to norepinephrine (beta(1)-, beta(2)-, beta(3)-, and alpha(2)-AR agonist), isoprenaline (beta(1)-, beta(2)-, and beta(3)-AR agonist), and CGP12177A (selective partial beta(3)-AR agonist but beta(1)- and beta(2)-AR antagonist) together with post-receptor agents, forskolin and dibutyryl cyclic AMP. The female rats that had greater oxygen consumption showed higher UCP1 content, a higher multilocular arrangement, and both longer cristae and higher cristae dense mitochondria in BAT indicating heightened thermogenic capacity and activity; this picture is accompanied by a more sensitive beta(3)-AR to norepinephrine signal (EC(50) 10-fold lower for CGP12177A) and a lower expression of alpha(2A)-AR than male rats. Taken together, our results support the idea that the BAT hormonal environment could be involved in the control of different elements of lipolytic and thermogenic adrenergic pathways. Gender dimorphism is both at receptor (changing alpha(2A)-AR density and beta(3)-AR affinity) and post-receptor (modulating the links involved in the adrenergic signal transduction) levels. These changes in adrenergic control could be responsible, at least in part, both for the important mitochondrial recruitment differences and functional and morphological features of BAT in female rats under usual rodent housing temperatures.     

Inhibition of dibutyryl cyclic AMP-induced insulin release by alpha-2 adrenergic stimulation

Effects of alpha adrenergic agonists and antagonists on dibutyryl cAMP (Bt2cAMP)-induced insulin release were investigated with isolated rat pancreatic islets. Bt2cAMP (4 mM) produced 2-fold stimulation of insulin release in all the concentrations of glucose examined (3.3-16.7 mM). Clonidine but not phenylephrine inhibited the Bt2cAMP-stimulated insulin release in a concentration-dependent manner with approximate EC50 of nanomolar range. Yohimbine but not prazosin antagonized the inhibitory effect of clonidine on the Bt2-cAMP-induced insulin release. These results suggest that alpha-2 adrenergic mechanisms are involved in a step distal to cAMP generation.     

Typing of the alpha adrenergic receptors of pancreatic B cells. In vitro studies with the isolated and perfused rat pancreas

Two groups of receptors, one which develops a stimulating effect (alpha 1), the other an inhibitory effect hae recently been isolated in the alpha adrenergic group. In order to type the B cell adrenergic receptor of the endocrine pancreas, which mediates the inhibitory action exerted by the catecholamines on insulin secretion, the release of this hormone was evaluated in the presence of five alpha simpathomimetic substances that have a decreasing degree of efficiency on the adrenergic alpha 2 receptor of the presynaptic sympathic nerve terminal. The order of potency with which the alpha agonists tested depressed IRI secretion is superimposable on that of their potency on the sympathetic nerve and alpha 2 receptor. We concluded that adrenergic inhibition of insulin secretion is mediated by an alpha 2 receptor.     

Contractile responses to adrenergic nerve stimulation are enhanced with removal of endothelium in rat caudal artery

Removal of the endothelium from isolated perfused rat caudal arteries produced a two fold increase in the contractile response to transmural nerve stimulation. Pretreatment with 6-hydroxydopamine eliminated the contractile response to adrenergic nerve stimulation but failed to uncover any vasodilatory effect of electrical stimulation, either directly on smooth muscle or via non-adrenergic nerves. Endothelial removal also produced two and four fold enhancement of the contractile responses to the selective alpha 1- and alpha 2-adrenoceptor agonists methoxamine and B-HT 920. However, pKB values for prazosin and yohimbine versus both agonists indicate that both methoxamine and B-HT 920 are acting primarily at alpha 1-adrenoceptors in this tissue. These results provide evidence that endothelial factors released either at basal levels or by the stimulation of agonists play a significant physiological role in modifying the contractile responses of blood vessels.     

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.     

Characterization of adrenergic receptors on proximal tubular basolateral membranes

Alpha adrenergic receptors are identified on basolateral plasma membranes derived from proximal tubular epithelial cells. The density of alpha 2 receptors was over two-fold greater than alpha 1 receptors. The basolateral membranes were devoid of beta receptors. These results support previous demonstrations of alpha adrenergic receptors in rat renal cortex and concur with studies which suggest a limited presence of beta receptors on rat proximal tubules.     

Limb-specific differences in the skin vascular responsiveness to adrenergic agonists

In this study, to test the hypothesis that adrenergic vasoconstrictor responses of the legs are greater compared with the arms in human skin, cutaneous vascular conductance (CVC) in the forearm and calf were compared during the infusion of adrenergic agonists in healthy young volunteers. Under normothermic conditions, norepinephrine (NE, α- and β-agonist, 1 × 10(-8) to 1 × 10(-2) M), phenylephrine (PHE, α(1)-agonist, 1 × 10(-8) to 1 × 10(-2) M), dexmedetomidine (DEX, α(2)-agonist, 1 × 10(-9) to 1 × 10(-4) M), and isoproterenol (ISO, β-agonist, 1 × 10(-8) to 1 × 10(-3) M) were administered by intradermal microdialysis. Skin blood flow (SkBF) was measured by laser-Doppler flowmetry, and the local temperature at SkBF-measuring sites was maintained at 34°C throughout the experiments. CVC was calculated as the ratio of SkBF to blood pressure and expressed relative to the baseline value before drug infusion. The dose of NE at the onset of vasoconstriction and the effective dose (ED(50)) resulting in 50% of the maximal vasoconstrictor response for NE were lower (P < 0.001) in the calf than forearm. The ED(50) for PHE and DEX was also lower (P < 0.05) in the calf than forearm. Increases in CVC in response to ISO were potentially smaller in the calf, but the statistical differences in the responses were dependent on the expressions of CVC. These findings suggest that the cutaneous vasoconstrictor responsiveness to exogenous NE is greater in the legs than in the arms due to a higher α(1)- and α(2)-adrenoceptor reactivity, while the β-adrenoceptor function plays a minor role in regional differences in adrenergic vasoconstriction in normothermic humans.     

Alpha adrenergic receptor mediated formation of cyclic AMP in rat spinal cord

Methoxamine and phenylephrine (PE), postsynaptic alpha adrenergic agonists stimulated the accumulation of cyclic AMP in spinal cord tissue slices. Naphazoline, oxymetazoline and clonidine, previously shown to have greater efficacy at presynaptic alpha receptors did not alter accumulation and, in fact, blocked the PE response. The PE-stimulation was completely inhibited by postsynaptic alpha antagonists, incompletely by agents which bl ock presynaptic alpha receptors, and slightly by the beta blocker propranolol. Pe-stimulated accumulation was potentiated by phosphodiesterase inhibition (RO 20-1724). In contrast to previous reports on the requirement of the copresence of adenosine for alpha receptor stimulated accumulation of cyclic AMP in neuronal tissue, the PE-stimulation in spinal cord slices was unchanged by adenosine receptor blockade (theophylline), hydrolysis of endogenous adenosine (adenosine deaminase), inhibition of adenosine deaminase (EHNA) or blockade of adenosine uptake (dipyridamole). Added adenosine increased basal accumulation and produced a marked potentiation of the PE response. From this data it is evident that, in spinal cord tissue slices, there occurs a postsynaptic alpha adrenergic receptor linked to cyclic AMP accumulation which does not require the presence of other neurohumoral agents for activation.