In vivo modulation by alpha 2-adrenoceptors of adrenal catecholamine release in the anaesthetized dog

In this study, the reversal of the potentiating effect of idazoxan, a selective alpha 2-antagonist, on adrenal catecholamine release elicited by splanchnic nerve stimulation in anaesthetized and vagotomized dogs, was investigated with the use of oxymetazoline, a selective alpha 2-agonist. Stimulation of the left splanchnic nerve (5.0-V pulses of 2 ms duration for 3 min at a frequency of 2 Hz) was applied before and 20 min after the i.v. injection of each drug. Blood samples were collected in the adrenal vein before and at the end of each stimulation. The results show that the release of catecholamines induced by electrical stimulation was potentiated by 50% after idazoxan injection (0.1 mg/kg). This enhanced response was significantly antagonized by the subsequent injection of oxymetazoline (2 micrograms/kg). The alpha 2-modulating effect appears to be related to the amount of catecholamines released during the stimulation, since by subgrouping of the data on the basis of the degree of potentiation by idazoxan, it was observed that this drug was more efficient when catecholamine release was higher during control stimulation. In contrast, the reversing effect of oxymetazoline was found to be more pronounced when catecholamine release was lower. These results thus suggest that the sensitivity of the alpha 2-adrenoceptor mechanism may depend upon the in situ concentration of adrenal catecholamine release during electrical stimulation and that the potentiating effect of alpha 2-blockade can be reversed by activation of those receptors by a selective alpha 2-agonist.     

The release of catecholamines from the adrenal medulla and its modulation by alpha 2-adrenoceptors in the anaesthetized dog

The adrenal nerve of anaesthetized and vagotomized dogs was electrically stimulated (10 V pulses of 2 ms duration for 10 min) at frequencies of 1, 3, 10, and 25 Hz. There was a correlation between the frequency of stimulation and the plasma concentrations of epinephrine, norepinephrine, and dopamine in the adrenal vein, mainly after the 1st min of stimulation and the maximal concentration was reached sooner with higher frequencies of stimulation. Moreover, the relative percentage of catecholamines released in response to the electrical stimulation was not changed by the frequency of stimulation. To test the hypothesis that a local negative feedback mechanism mediated by alpha 2-adrenoceptors exists in the adrenal medulla, the effects of the systemic administration of clonidine (alpha 2-antagonist) on the concentrations of catecholamines in the adrenal vein were evaluated during the electrical stimulation of the adrenal nerve (5 V pulses of 2 ms duration for 3 min) at 3 Hz. Moreover, the effects of the systemic injections of more specific alpha 2-agonist and antagonist (oxymetazoline and idazoxan) were tested on the release of catecholamines in the adrenal vein in response to electrical stimulation of the splanchnic nerve at 1 and 3 Hz frequencies. The injection of 0.5 mg/kg of yohimbine caused a significant increase in the concentrations of epinephrine and norepinephrine in the adrenal vein induced by the electrical stimulation of the adrenal nerve and the injection of 15 micrograms/kg of clonidine had no effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between angiotensin III and alpha 2-adrenoceptors of the medulla oblongata involved in cardiovascular regulation in the rat

We evaluated, in adult, male Sprague-Dawley rats anesthetized with pentobarbital sodium, possible interaction between angiotensin III (AIII) and the alpha 2-adrenoceptors in the medulla oblongata that are involved in cardiovascular regulation. The hypotensive and negative chronotropic and inotropic actions of the alpha 2-adrenoceptor agonist, guanabenz, were used as our experimental index. Intracerebroventricular (i.c.v.) administration of AIII (100 or 200 pmol) significantly attenuated the cardiovascular suppressive effects of the aminoguanidine compound (25 or 50 micrograms/kg, i.v.). Bilateral microinjection of AIII (20 or 40 pmol) to the nucleus reticularis gigantocellularis (NRGC), a medullary site believed to be intimately related to the antihypertensive action of guanabenz, produced similar results. In addition, i.c.v. administered AIII (200 pmol) altered the effects of guanabenz on the arterial pressure-related neurons in the NRGC, in a manner that paralleled the blunted vasodepressive action of the aminoguanidine compound by the heptapeptide. When applied microiontophoretically, AIII also significantly decreased the responsiveness of arterial pressure-related neurons in the NRGC to guanabenz. These findings suggest that AIII may interact with the alpha 2-adrenoceptors located in the NRGC that are involved in central cardiovascular regulation.     

Metabolic regulation by alpha 1- and alpha 2-adrenoceptors.

The role of alpha-adrenoceptors in the mediation of autonomic function, particularly in the control of the cardiovascular system, is widely known. However, alpha-adrenoceptors are also important in the regulation of a variety of metabolic processes that occur in the body either through direct action or by stimulation of the release of other mediators that control metabolic function. Thus, alpha 2-adrenoceptor activation by circulating or neuronally released catecholamines inhibits the release of insulin from pancreatic islet beta-cells and, by inhibiting this response, alpha 2-adrenoceptor antagonists have been shown to have an antihyperglycemic effect. The alpha-adrenoceptor-mediated regulation of the release of pituitary hormones is indirect, with alpha-adrenoceptors being located on peptidergic neurons in the hypothalamus that secrete releasing hormones into the hypophysial portal system to regulate the secretion of hormones from the anterior pituitary gland. Thus, the increase in cortisol secretion from the adrenal glands following a meal is produced, at least in part, by an alpha 1-adrenoceptor-mediated increase in vasopressin and CRF-41 secretion from neurons on the hypothalamus that stimulate the release of adrenocorticotrophic hormone secretion from the pituitary gland, which subsequently stimulates the synthesis and release of cortisol from the adrenal medulla. In addition to metabolic regulation by alpha 1- and alpha 2-adrenoceptors within the endocrine system, alpha-adrenoceptors are also a component of the system that regulates certain aspects of metabolism within autonomic effector cells, such as the control of smooth muscle cell division and growth during periods of continued alpha-adrenoceptor activation as a result of activation of second messenger systems.     

Presynaptic mediation by alpha 2-, beta 1- and beta 2-adrenoceptors of endogenous noradrenaline and dopamine release from slices of rat hypothalamus

Using high performance liquid chromatography with an electro-chemical detector, we studied effects of different compounds on the impulse-evoked release of endogenous noradrenaline (NA) and dopamine (DA) release from slices of the rat hypothalamus. Adrenaline (10(-7) M), with a potent alpha-agonistic action decreased both NA and DA release, and these effects were blocked by pretreatment with yohimbine (10(-7 M). The alpha 2-antagonist, yohimbine alone (10(-8) - 10(-6) M) concentration-dependently increased these releases, while alpha 1-antagonist, prazosin showed weak increase on NA but not DA release at 10(-6) M. Isoproterenol (10(-10) - 10(-8) M) concentration-dependently increased these releases and the effects were antagonized by pretreatment with a non-selective beta-antagonist, 1-propranolol, a beta 1-antagonist, atenolol or a beta 2-antagonist, butoxamine. 1-Propranolol (3 X 10(-7) M) alone, but not the d-isomer inhibited the releases. Thus, in the rat hypothalamus, the release of NA and DA may be mediated via presynaptic alpha 2-, beta 1- and beta 2-adrenoceptors.     

M2 muscarinoceptor-associated ionophore at the cat adrenal medulla

Atropine and pirenzepine displaced 3H-quinuclydinyl-benzylate binding and inhibited methacholine-evoked catecholamine release with a similar order of potencies, atropine being 200 fold more potent than pirenzepine. In contrast to high-K, methacholine-evoked 45Ca uptake or catecholamine release were not blocked by (+)PN200-110. Bay-K-8644 did not modify the secretory response to methacholine either in the presence of Ca or Sr but potentiated K-evoked secretion. In depolarized glands, methacholine still evoked its usual secretory response. The results suggest that muscarinic stimulation of cat adrenal chromaffin cells stimulates Ca entry though an ionophore other than voltage-dependent Ca channels; such ionophore seems to be chemically operated through a M2 muscarinoceptor.     

The interaction of the enantiomers of carvedilol with alpha 1- and beta 1-adrenoceptors

The stereoselectivity of carvedilol, a novel beta-adrenoceptor antagonist and vasodilator with one asymmetric carbon atom, was examined at alpha 1- and beta 1-adrenoceptors in vitro and in vivo. (-)-(S)-Carvedilol is a potent, competitive antagonist of the beta 1-adrenoceptor-mediated positive chronotropic response to isoproterenol in guinea pig atrium, with a dissociation constant (KB) of 0.4 nM. (+)-(R)-Carvedilol was more than 100-fold less potent than the (-)-S-enantiomer as an antagonist of beta 1-andrenoceptors, having a KB of approximately 45 nM. Consistent with these findings (-)-(S)-carvedilol (0.1 mg/kg, i.v.) produced a 25-fold rightward shift in the beta 1-adrenoceptor-mediated positive chronotropic response to isoproterenol in pithed rats, whereas the (+)-R-enantiomer had no beta 1-adrenoceptor blocking activity in vivo at this dose. In contrast to the marked degree of stereoselectivity observed at beta 1-adrenoceptors, both (-)-(S)- and (+)-(R)-carvedilol produced equal antagonism of the alpha 1-adrenoceptor-mediated vasoconstrictor response to norepinephrine in rabbit aorta, with KB values of 14 and 16 nM, respectively. Furthermore, in the pithed rat, the alpha 1-adrenoceptor-mediated pressor dose-response curve to cirazoline was shifted approximately 6-fold to the right by both the (+)-R- and (-)-S-enantiomers of carvedilol at a dose of 1 mg/kg, i.v. In anesthetized spontaneously hypertensive rats, (-)-(S)-carvedilol was 6-fold more potent as an antihypertensive than (+)-(R)-carvedilol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic modulation of transmitter release via alpha2-adrenoceptors: nonsynaptic interactions

It is generally accepted that neurochemical transmission occurring at the synapse is the primary way of sending messages from one neuron to another. Neurotransmitters released from axon terminal in a [Ca2+]0-dependent manner act transsynaptically on the postsynaptic site. The past 30 years have witnessed something of a revolution in the understanding of how neurons communicate with each other. It has been shown that the exocytotic release of transmitters from axon terminals is subject to presynaptic modulation via presynatic hetero- and auto-receptors. For example via stimulation of alpha2-adrenoceptors expressed on varicosities and coupled to G-protein the stimulation-evoked release of different transmitters can be inhibited. This review will focus on nonsynaptic interactions between axon terminals. The present data clearly show that transmitters released from axon terminals without synaptic contact play an important role in the fine tuning of communication between neurons within a neuronal circuit.     

Calcium handling in vasoconstriction to stimulation of alpha 1- and alpha 2-adrenoceptors

Vasoconstriction to stimulation of postsynaptic alpha 1- and alpha 2-adrenoceptors involves different mechanisms of Ca2+ mobilization. Alpha 2-adrenoceptor-mediated vasoconstriction in vivo as well as in vitro is invariably and effectively antagonized by Ca2+ channel blockers, such as nifedipine or verapamil, and is therefore primarily carried by influx of extracellular Ca2+. On the other hand, alpha 1-adrenoceptor stimulation has been linked to both influx of extracellular Ca2+ and release of Ca2+ from intracellular stores. The sensitivity of alpha 1-adrenoceptor-mediated vasoconstriction to blockade by Ca2+ channel antagonists depends on how much both mechanisms of Ca2+ mobilization contribute to the contraction process, and varies between vascular tissues and alpha 1-adrenoceptor agonists. The experimental evidence for the differential utilization of Ca2+ in vasoconstriction to alpha 1- and alpha 2-adrenoceptor stimulation is reviewed.     

Coexistence of beta 1- and beta 2-adrenoceptors in the melanophore of the goby Tridentiger obscurus

The effects of beta-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the beta-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these beta-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both alpha- and beta-adrenoceptors, and that the activation of the beta-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two beta 1-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of beta 1-receptors. By contrast, the pigment-dispersing effects of two beta 2-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the beta-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest co-existence of beta 1- and beta 2-adrenoceptors in the melanophores. The relative numbers of beta 1- and beta 2-adrenoreceptors as a percentage of the total population of beta-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the beta-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit interfaces

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.     

Mechanism of synaptic inhibition by noradrenaline acting at alpha 2-adrenoceptors

The actions of agonists at alpha 2-adrenoceptors were investigated on single cells of the submucous plexus of the guinea pig small intestine. Intracellular recordings were made from neurons in vitro, and noradrenaline and other agonists were applied by adding them to the superfusion solution. The actions of noradrenaline released from terminals of sympathetic nerves was also studied by stimulating the nerves and recording the inhibitory postsynaptic current; this current can be mimicked by brief applications of noradrenaline from a pipette tip positioned within 50 micron of the neuron. The alpha 2-adrenoceptor-bound noradrenaline with an apparent dissociation constant of 15 microM, determined by the method of partial irreversible receptor inactivation: clonidine and 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304) had dissociation constants of 36 nM and 2.5 microM respectively. Noradrenaline and UK 14304 caused maximal hyperpolarizations, or outward currents; clonidine was a full agonist in only 4 of 35 cells, a partial agonist in 25 cells, and without effect in 4 cells. Clonidine acted as a competitive antagonist of noradrenaline in those cells in which it lacked agonist action; its dissociation equilibrium constant determined by Schild analysis was about 20 nM. The potassium conductance increased by the alpha 2-adrenoceptor agonists, whether they were applied exogenously or released by stimulation of presynaptic nerves, showed marked inward rectification. The neurons showed inward rectification also in the absence of agonist; both types of rectification were eliminated by rubidium (2 mM), barium (3-30 microM) and caesium (2 mM). When the recording electrodes contained the nonhydrolysable derivative of guanosine 5'-triphosphate (GTP), guanosine 5'-O-(3-thiotriphosphate, GTP-gamma-S), the effects of applied alpha 2-adrenoceptor agonists did not reverse when they were washed from the tissue, implying that GTP hydrolysis is necessary for the termination of agonist action. Pretreatment with pertussis toxin abolished the inhibitory synaptic potential (IPSP) and agonist-induced hyperpolarizations. Phorbol 12,13-dibutyrate, forskolin, cholera toxin and sodium fluoride did not affect the responses to alpha 2-adrenoceptor agonists. The synaptic hyperpolarization resulting from sympathetic nerve stimulation, or the hyperpolarization evoked by a brief (3-5 ms) application of noradrenaline, began after a latency of about 30 and 60 ms respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decrease in rat cardiac beta 1- and beta 2-adrenoceptors by training and endurance exercise

The cardiac beta-adrenoceptor adaptation to physical activity was investigated in rats which were subjected to a six-week endurance swimming training (ET; n = 7) and a training of high intensity (MT; n = 7). In addition, the effect of a single bout of endurance exercise without preceding training (EE; n = 7) was evaluated. These groups were compared with a sedentary control group (C; n = 9). Beta-adrenergic receptors in rat myocardial membranes were labelled using the high affinity antagonist radioligand (-)125iodocyanopindolol (ICYP). Computer modelling techniques provided estimates of the maximal binding capacity (Bmax) and the dissociation constants (KD). Tissue was constantly kept at temperatures of less than or equal to 4 degrees C and incubated at 4 degrees C for 18 h in buffer containing 100 microM GTP so as to prevent masking of beta-adrenoceptors by endogenous norepinephrine. In comparison with the C group (Bmax = 43.2 +/- 1.6 fmol/mg protein, KD = 11.7 +/- 1.5 pM) computerized coanalyses of saturation binding data of ET, MT, and EE revealed a 13.0%, 25.5%, and 16.6% decrease in Bmax (P less than 0.01), respectively, without significantly differing KD values (10.6 pM, 9.0 pM, 10.5 pM, respectively). We provide the first evidence that acute exercise lowers the sarcolemmal beta-adrenoceptor number in the rat heart. In the competition radioligand binding, CGP20712A and ICI118.551 were employed as subtype-selective antagonists of beta 1- and beta 2-adrenoceptors, respectively, to determine the relative proportions of the receptor subtypes. The ratio of beta 1-/beta 2-adrenoceptors in C was 67.5:32.5 and no statistically significant variation occurred in animals subjected to physical activity. On the basis of published data we assume that acute exercise induces a sequestration of beta-adrenoceptors from the cell surface to some intracellular compartment, whereas the molecular basis of the chronic beta-adrenoceptor down-regulation may involve a training-induced reduction in receptor synthesis. Our findings on cardiac beta-adrenoceptor adaptation to physical activity may represent one of the mechanisms underlying the relative bradycardia in trained subjects.     

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.     

In vivo imaging of molecular interactions at damaged sarcolemma

Muscle cells have a remarkable capability to repair plasma membrane lesions. Mutations in dysferlin (dysf) are known to elicit a progressive myopathy in humans, probably due to impaired sarcolemmal repair. We show here that loss of Dysf and annexin A6 (Anxa6) function lead to myopathy in zebrafish. By use of high-resolution imaging of myofibers in intact animals, we reveal sequential phases in sarcolemmal repair. Initially, membrane vesicles enriched in Dysf together with cytoplasmic Anxa6 form a tight patch at the lesion independently of one another. In the subsequent steps, annexin A2a (Anxa2a) followed by annexin A1a (Anxa1a) accumulate at the patch; the recruitment of these annexins depends on Dysf and Anxa6. Thus, sarcolemmal repair relies on the ordered assembly of a protein-membrane scaffold. Moreover, we provide several lines of evidence that the membrane for sarcolemmal repair is derived from a specialized plasma membrane compartment.     

Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.