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Fatty acid acylation of dopamine in the carotid body
Medical Hypotheses (1998)50, 131-133
© HarcourtBrace& Co. Ltd 1998
Fatty acid acylation of dopamine in the carotid body M. POKORSKI, Z. MATYSIAK* Medical Research Center and *Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland (Correspondence to: Dr M. Pokorski, Department of Neurophysiology, Medical Research Center, Polish Academy of Sciences, Dworkowa 3 St, 00-784 Warsaw, Poland. Fax: +48 22 495829; e-maih [email protected], waw.pl)
Abstract - - In this article, we put forward a hypothesis concerning the assembling and storage of dopamine molecules in the dense-core vesicles of the carotid body chemoreceptor cell. We posit that dopamine molecules are packed and sustained in the vesicular form due to the formation of N-acyldopamine, a condensation product of fatty acid acyl chain and dopamine at the amino group of the latter. N-acyldopamine would then be stored in a micelle-like supramolecular structure formed due to self-association through the hydrophilic dopamine headgroups. This hypothesis may help explain the perennial problem of the role of dopamine in chemoception. It also draws attention to the possibility of the existence of neurotransmitters in the N-acylated form. This could lead to the design of acylated compounds that would play a role of prodrugs slow-releasing active substances by hydrolysis into the desired environment.
Background The chemoreceptor cells of the carotid body sense reductions in arterial blood oxygen tension and transduce that information into increased afferent impulse traffic in the apposed nerve endings. This results in a reflex increase of the bralnstem respiratory motor output to meet the metabolic needs. The intramolecular mechanisms of transduction of the hypoxic stimulus are unclear. One of the few established tenets of the chemoreceptor cell function is the release of various neurotransmitters at the time of excitation. Of these, dopamine (DA) has attracted wide attention, being released from the chemoreceptor cell by both natural, like hypoxia, and phar-
macological, like cyanide, stimuli (1). DA is stored in the dense-core vesicles from which it released in a Ca2+-dependent, exocytotic way. The vesicles gather mostly in the outer boundary of the cytoplasm, where they often fuse. The role of DA in chemoception is a contentious issue. The reports are conflicting, showing both inhibitory and excitatory effects on both the chemoreceptor afferent discharge rate and respiration of DA, depending on the species, the dosage and the experimental paradigm employing various agonists and antagonists. One concept holds that these unsealed effects depend on a different affinity of pre- and postsynaptic D 2 receptor sites in the carotid body (2), which may also well be due to recently
Received 11 June 1996 Accepted23 October 131
13 2
MEDICAL HYPOTHESES
described subclasses of the D2 receptor. The nebulous role of DA may be the reason that the prevailing view of its importance in the chemoception cascade swayed back and forth over the years. The general consensus that DA is an inhibitory neurotransmitter in the chemoreceptor cell currently prevails.
Hypothesis We think the basic issue in understanding the action of DA involves the molecular mechanisms of its assembling and storage in the dense-core vesicles. In this paper we are putting forward a hypothesis in this regard. The question arises of how dopamine molecules are assembled into the dense-core vesicle milieu. The physicochemical properties and hydrophilicity of DA molecules make it unlikely that they could be packed and sustained in the vesicular entity. We hypothesize that the processing of DA molecules into the vesicular form is due to the structural perturbation of NH2-terminal of the molecule that allows its combination with the COOH-terminal of a fatty acid. N-acyldopamine is formed. This is a dopamine to the N of which an acyl group is attached according to the general formula:
H I R1
O
II N--C--R2
where R 1 is catecholamines and R 2 is fatty acids. The N-acyldopamine molecules would then form a micelle-like supramolecular structure due to selfassociation through the hydrophilic DA headgroups with the lipophilic fatty acid alkyl groups placed on the outer layer of the micelle. Incorporation of fatty acid alkyl tails into the micelle would increase its hydrophobic character. Such a structure would explain the sustenance of DA molecules within the confined vesicle, which would also be aided by the interfacial balance of the alkyl chain and the DA headgroup. Fatty acids of various length could provide the alkyl residues for combining with DA, which could result in a different size of micellar entities and underlie the known anisometric shape of the dense-core vesicles.
Supportive evidence Acylation of some intracellular constituents has been described previously and its potential importance is increasingly recognized. Cell membrane glycoproteins become acylated with fatty acids in the Golgi elements (3) on the way from the rough endoplasmic reticulum, where they are synthesized to the cell surface (4).
As far as we are aware, nothing is known about the intracellular acylation of dopamine. N-acyldopamines were synthesized from dopamine and fatty acids and reported to inhibit the in vitro synthesis of leukotrienes (5). The inhibition was greater with the unsaturated lipophilic alkyl groups of more than Clo. N-acyldopamine may thus have a biological activity. The chemoreceptor cell abounds with both catechol and unsaturated lipophilic alkyl groups and the condensation of the two does not seem implausible. Both are elaborated during the application of stimuli. Hypoxia releases DA (1), activates the metabolism of arachidonic acid (6), and increases the prostaglandin E2 level (7). The source of unsaturated fatty acids may be the hydrolysis of membrane phospholipids in hypoxia (8). Chromaffin granules of the adrenal gland, which are similar secretory sacs, release both epinephrine and arachidonic acid upon stimulation and extensive exocytosis that follows (9). The fusion of these secretory vesicles is markedly enhanced by the addition of a small amount of a cis-unsaturated fatty acid, like arachidonic acid. Moreover, studies with labeled arachidonic acid showed that nearly all of it was bound to the granule, and not incorporated into phospholipids, at the time of fusion. The mechanisms underlying these in vitro processes may be available in the cytoplasm of the chemoreceptor cell when it is stimulated. The acylation of membrane proteins described above and the presence of unsaturated fatty acids in both the chemoreceptor function and as fusogen strengthen the hypothesis of possible acylation of DA in the densecore vesicles in the chemoreceptor cell. Another hint of support comes from the experiments in which phenylmethylsulfonyl fluoride (PMSF), a toxic substance reacting with amino groups, induces widespread degenerative changes in all ultrastructural elements of the carotid body parenchyma but the dense-core vesicles (10). If DA had free amino groups, PMSF would likely clean them out and dissolve the vesicle. N-acyldopamine could either actively interact with the dopamine receptor or be a relatively stable precursor form with little biological activity which would be processed into active DA due to deacylation on stimulation. If the latter were true, than exogenous N-acyldopamine loaded into the chemoreceptor cell could dampen the inhibitory effect on its afferent discharge rate of DA. The biological activity of N-acyldopamine is yet to be tested.
Significance The hypothesis of neurotransmitters existing in the N-acylated form has implications reaching far beyond
FATrY ACIDACYLATIONOF DOPAMINEIN THE CAROTIDBODY
133
the function of DA in the carotid body. Such acylated compounds could play a role of prodrugs which would slow-release active substances by hydrolysis in a desired environment. Parkinson's disease may be a case in point in regard to DA. Lipophilic Nacyldopamine, as opposed to DA, could cross the blood-brain barrier and be the source of slowreleased DA, and therefore ameliorate the symptoms. Definitive conclusions should await the synthesis of the chemically acylated neurotransmitters in question, which would serve as standards for the biochemical identification of those postulated to exist in vivo and would also be the subject of studies of their biological actions.
Neurosci 1992; 15: 146-153. 2. Gonzalez C, Almaraz L, Obeso A, Rigual R. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol Rev 1994; 74: 829-898. 3. Schmidt M F G, Schlesinger M J. Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J Biol Chem 1980; 255: 3334-3339. 4. Schlesinger M J, Magee A I, Schmidt M F G. Fatty acid acylation of proteins in cultured cells. J Biol Chem 1980; 255: 10021-10024. 5. Tseng C-F, Iwakami S, Mikajiri A et al. Inhibition of in vitro prostaglandin and leukotriene biosynthesis by cinnamoyl-~phenethylamine and N-acyldopamine derivatives. Chem Pharm Bull 1992; 40: 396-400. 6. Strosznajder R P, Pokorski M. Hypoxia affects arachidonic acid incorporation into phospholipids in cat carotid bodies. Eur Respir J 1995; 8 Suppl. 19: 410s, P2030. 7. Gomez-Nino A, Almaraz L, Gonzalez C. In vitro activation of cyclo-oxygenase in the rabbit carotid body by natural stimuli. J Physiol London 1994; 476: 257-267. 8. Pokorski M, Strosznajder R. PO2-dependence of phospholipase C in the cat carotid body. Adv Exp Med Biol 1993; 337: 191-195. 9. Creutz C E, Pollard H B. A cell-free model for proteinlipid interactions in exocytosis. Aggregation and fusion of chromaffin granules in the presence of calcium, synexin, and cis-unsaturated fatty acids. Biophys J 1982; 37:119-120. 10. Pokorski M, Walski M, Matysiak Z. A phospholipase C inhibitor impedes the hypoxic ventilatory response in the cat. Adv Exp Med Biol 1996; 410: 397-403.
Acknowledgement This research was supported by the statutory budget of the Polish Academy of Sciences.
References 1. Gonzalez C, Almaraz L, Obeso A, Rigual R. Oxygen and acid chemoreception in the carotid body chemoreceptors. Trends
© HarcourtBrace& Co. Ltd 1998
Fatty acid acylation of dopamine in the carotid body M. POKORSKI, Z. MATYSIAK* Medical Research Center and *Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland (Correspondence to: Dr M. Pokorski, Department of Neurophysiology, Medical Research Center, Polish Academy of Sciences, Dworkowa 3 St, 00-784 Warsaw, Poland. Fax: +48 22 495829; e-maih [email protected], waw.pl)
Abstract - - In this article, we put forward a hypothesis concerning the assembling and storage of dopamine molecules in the dense-core vesicles of the carotid body chemoreceptor cell. We posit that dopamine molecules are packed and sustained in the vesicular form due to the formation of N-acyldopamine, a condensation product of fatty acid acyl chain and dopamine at the amino group of the latter. N-acyldopamine would then be stored in a micelle-like supramolecular structure formed due to self-association through the hydrophilic dopamine headgroups. This hypothesis may help explain the perennial problem of the role of dopamine in chemoception. It also draws attention to the possibility of the existence of neurotransmitters in the N-acylated form. This could lead to the design of acylated compounds that would play a role of prodrugs slow-releasing active substances by hydrolysis into the desired environment.
Background The chemoreceptor cells of the carotid body sense reductions in arterial blood oxygen tension and transduce that information into increased afferent impulse traffic in the apposed nerve endings. This results in a reflex increase of the bralnstem respiratory motor output to meet the metabolic needs. The intramolecular mechanisms of transduction of the hypoxic stimulus are unclear. One of the few established tenets of the chemoreceptor cell function is the release of various neurotransmitters at the time of excitation. Of these, dopamine (DA) has attracted wide attention, being released from the chemoreceptor cell by both natural, like hypoxia, and phar-
macological, like cyanide, stimuli (1). DA is stored in the dense-core vesicles from which it released in a Ca2+-dependent, exocytotic way. The vesicles gather mostly in the outer boundary of the cytoplasm, where they often fuse. The role of DA in chemoception is a contentious issue. The reports are conflicting, showing both inhibitory and excitatory effects on both the chemoreceptor afferent discharge rate and respiration of DA, depending on the species, the dosage and the experimental paradigm employing various agonists and antagonists. One concept holds that these unsealed effects depend on a different affinity of pre- and postsynaptic D 2 receptor sites in the carotid body (2), which may also well be due to recently
Received 11 June 1996 Accepted23 October 131
13 2
MEDICAL HYPOTHESES
described subclasses of the D2 receptor. The nebulous role of DA may be the reason that the prevailing view of its importance in the chemoception cascade swayed back and forth over the years. The general consensus that DA is an inhibitory neurotransmitter in the chemoreceptor cell currently prevails.
Hypothesis We think the basic issue in understanding the action of DA involves the molecular mechanisms of its assembling and storage in the dense-core vesicles. In this paper we are putting forward a hypothesis in this regard. The question arises of how dopamine molecules are assembled into the dense-core vesicle milieu. The physicochemical properties and hydrophilicity of DA molecules make it unlikely that they could be packed and sustained in the vesicular entity. We hypothesize that the processing of DA molecules into the vesicular form is due to the structural perturbation of NH2-terminal of the molecule that allows its combination with the COOH-terminal of a fatty acid. N-acyldopamine is formed. This is a dopamine to the N of which an acyl group is attached according to the general formula:
H I R1
O
II N--C--R2
where R 1 is catecholamines and R 2 is fatty acids. The N-acyldopamine molecules would then form a micelle-like supramolecular structure due to selfassociation through the hydrophilic DA headgroups with the lipophilic fatty acid alkyl groups placed on the outer layer of the micelle. Incorporation of fatty acid alkyl tails into the micelle would increase its hydrophobic character. Such a structure would explain the sustenance of DA molecules within the confined vesicle, which would also be aided by the interfacial balance of the alkyl chain and the DA headgroup. Fatty acids of various length could provide the alkyl residues for combining with DA, which could result in a different size of micellar entities and underlie the known anisometric shape of the dense-core vesicles.
Supportive evidence Acylation of some intracellular constituents has been described previously and its potential importance is increasingly recognized. Cell membrane glycoproteins become acylated with fatty acids in the Golgi elements (3) on the way from the rough endoplasmic reticulum, where they are synthesized to the cell surface (4).
As far as we are aware, nothing is known about the intracellular acylation of dopamine. N-acyldopamines were synthesized from dopamine and fatty acids and reported to inhibit the in vitro synthesis of leukotrienes (5). The inhibition was greater with the unsaturated lipophilic alkyl groups of more than Clo. N-acyldopamine may thus have a biological activity. The chemoreceptor cell abounds with both catechol and unsaturated lipophilic alkyl groups and the condensation of the two does not seem implausible. Both are elaborated during the application of stimuli. Hypoxia releases DA (1), activates the metabolism of arachidonic acid (6), and increases the prostaglandin E2 level (7). The source of unsaturated fatty acids may be the hydrolysis of membrane phospholipids in hypoxia (8). Chromaffin granules of the adrenal gland, which are similar secretory sacs, release both epinephrine and arachidonic acid upon stimulation and extensive exocytosis that follows (9). The fusion of these secretory vesicles is markedly enhanced by the addition of a small amount of a cis-unsaturated fatty acid, like arachidonic acid. Moreover, studies with labeled arachidonic acid showed that nearly all of it was bound to the granule, and not incorporated into phospholipids, at the time of fusion. The mechanisms underlying these in vitro processes may be available in the cytoplasm of the chemoreceptor cell when it is stimulated. The acylation of membrane proteins described above and the presence of unsaturated fatty acids in both the chemoreceptor function and as fusogen strengthen the hypothesis of possible acylation of DA in the densecore vesicles in the chemoreceptor cell. Another hint of support comes from the experiments in which phenylmethylsulfonyl fluoride (PMSF), a toxic substance reacting with amino groups, induces widespread degenerative changes in all ultrastructural elements of the carotid body parenchyma but the dense-core vesicles (10). If DA had free amino groups, PMSF would likely clean them out and dissolve the vesicle. N-acyldopamine could either actively interact with the dopamine receptor or be a relatively stable precursor form with little biological activity which would be processed into active DA due to deacylation on stimulation. If the latter were true, than exogenous N-acyldopamine loaded into the chemoreceptor cell could dampen the inhibitory effect on its afferent discharge rate of DA. The biological activity of N-acyldopamine is yet to be tested.
Significance The hypothesis of neurotransmitters existing in the N-acylated form has implications reaching far beyond
FATrY ACIDACYLATIONOF DOPAMINEIN THE CAROTIDBODY
133
the function of DA in the carotid body. Such acylated compounds could play a role of prodrugs which would slow-release active substances by hydrolysis in a desired environment. Parkinson's disease may be a case in point in regard to DA. Lipophilic Nacyldopamine, as opposed to DA, could cross the blood-brain barrier and be the source of slowreleased DA, and therefore ameliorate the symptoms. Definitive conclusions should await the synthesis of the chemically acylated neurotransmitters in question, which would serve as standards for the biochemical identification of those postulated to exist in vivo and would also be the subject of studies of their biological actions.
Neurosci 1992; 15: 146-153. 2. Gonzalez C, Almaraz L, Obeso A, Rigual R. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol Rev 1994; 74: 829-898. 3. Schmidt M F G, Schlesinger M J. Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J Biol Chem 1980; 255: 3334-3339. 4. Schlesinger M J, Magee A I, Schmidt M F G. Fatty acid acylation of proteins in cultured cells. J Biol Chem 1980; 255: 10021-10024. 5. Tseng C-F, Iwakami S, Mikajiri A et al. Inhibition of in vitro prostaglandin and leukotriene biosynthesis by cinnamoyl-~phenethylamine and N-acyldopamine derivatives. Chem Pharm Bull 1992; 40: 396-400. 6. Strosznajder R P, Pokorski M. Hypoxia affects arachidonic acid incorporation into phospholipids in cat carotid bodies. Eur Respir J 1995; 8 Suppl. 19: 410s, P2030. 7. Gomez-Nino A, Almaraz L, Gonzalez C. In vitro activation of cyclo-oxygenase in the rabbit carotid body by natural stimuli. J Physiol London 1994; 476: 257-267. 8. Pokorski M, Strosznajder R. PO2-dependence of phospholipase C in the cat carotid body. Adv Exp Med Biol 1993; 337: 191-195. 9. Creutz C E, Pollard H B. A cell-free model for proteinlipid interactions in exocytosis. Aggregation and fusion of chromaffin granules in the presence of calcium, synexin, and cis-unsaturated fatty acids. Biophys J 1982; 37:119-120. 10. Pokorski M, Walski M, Matysiak Z. A phospholipase C inhibitor impedes the hypoxic ventilatory response in the cat. Adv Exp Med Biol 1996; 410: 397-403.
Acknowledgement This research was supported by the statutory budget of the Polish Academy of Sciences.
References 1. Gonzalez C, Almaraz L, Obeso A, Rigual R. Oxygen and acid chemoreception in the carotid body chemoreceptors. Trends