(Regitine, Ciba), propranolol hydrochloride (Inderal, Ayerst), and hexamethonium.

Transcription

1 STIMULATION OF THE CAROTID CHEMORECEPTORS OF THE DOG BY DOPAMIINE* BY LOUISE JACOBS AND JULIUS H. COMROE, JR. CARDIOVASCULAR RESEARCH INSTITUTE, UNIVERSITY OF CALIFORNIA SCHOOL OF MEDICINE, SAN FRANCISCO Communicated January 29, 1968 The mechanisms by which chemosensitive receptors in the carotid body are activated by the changes in the Po2, Pco,, or ph of arterial blood or by a variety of chemicals are still unknown. Physiological activation may require the release of a transmitter substance, formed or stored in Type I chemoreceptor cells, which then excites contiguous sensory nerve endings. Recently, a number of investigators, using the techniques of bioassay, fluorescence microscopy, or separation and chemical analysis, have found that the carotid body contains acetylcholine, serotonin, and three catecholamines (epinephrine, norepinephrine, and dopamine).1-v Of these, only dopamine has not been studied for its physiological effects on the chemoreceptors. t Because of the widespread belief that dopamine may play an important role in activating neurons in certain regions of the brain, we investigated the effects of dopamine on the sensory receptors of the carotid body. We found it to be a potent stimulus. Materials and Al ethods.-we used 15 dogs, 5-22 kg, which were anesthetized by alpha chloralose ( mg/kg i.v.) prepared as a 10% solution in polyethylene glycol 200. In order to inject drugs close to the carotid body, we inserted a fine polyethylene catheter into the lingual artery and pushed it toward the heart until it reached the common carotid artery just before it bifurcates. We ligated the internal carotid artery, the ascending pharyngeal artery, and the external carotid artery beyond the carotid body, leaving patent only the occipital artery, which provides the blood supply to the carotid body. This procedure decreases blood flow through that common carotid artery so that small amounts of intraarterially injected drugs will produce concentrations in the carotid blood high enough to stimulate the carotid body without producing systemic effects upon recirculation. The actual concentration of the drug in the carotid arterial blood depends not only on the amount injected and the rate of injection but also on the carotid blood flow; the last varies with the size of the dog, the carotid blood pressure, and the peripheral resistance in the occipital arterioles. A plastic tube was tied in a femoral vein for intravenous injections. In some dogs, a no. 7 Cournand catheter was passed down an external jugular vein into the right atrium or ventricle for injection of drugs into the right heart. The chemicals used were: sodium cyanide, nicotine bitartrate, dopamine, i-tyrosine, L-DOPA, norepinephrine (Levophed, 3 Winthrop), homovanillic acid, phentolamine (Regitine, Ciba), propranolol hydrochloride (Inderal, Ayerst), and hexamethonium. All drugs were dissolved in 0.9% saline except for Irtyrosine and L-DOPA, which required the addition of 1 N NaOH (final ph 10.3). The norepinephrine solution contained 0.2% sodium bisulfite as a preservative. Intracarotid injections of dilute NaOH (ph 10.5) or 0.2% sodium bisulfite alone had no effect. The dogs breathed spontaneously through a tracheal cannula and a Fleisch pneumotachograph. To measure arterial blood pressure, we used a Statham strain gauge connected to a catheter placed in a femoral artery. A Grass polygraph recorded arterial blood pressure, rate of inspiratory and expiratory air flow, tidal volume (by electrical integration of air flow), and the percentage of CO2 in inspired and expired gas (measured by a Beckman LB 1 CO2 meter). 1187

2 1188 PHYSIOLOGY: JACOBS AND COMROE PROC. N. A. S. Results.-In each of the 15 dogs, intracarotid injections of dopamine produced the abrupt, intense, and brief hyperpnea that is characteristic of carotid-body stimulation (Fig. 1). The frequency and tidal volume of respiration increased and the Pco, of end-tidal expired air decreased. In most instances the increase of the tidal volume was greatest during the first breath after the injection. The response increased as the dose increased, and there was no evidence of tachyphylaxis. The effects of dopamine on the circulation were similar to those elicited by other chemical agents that stimulate the carotid bodies (e.g., nicotine and cyanide): there was either no effect or a transient bradycardia and hypotension. These respiratory and circulatory effects were due to carotid-body reflexes be- % C02 2 G _ 'IIi 'A i RESP 9 / FLOW 6 r L /sec 0--l r, -.-,IJ III I ; I I 1 I 600!- TIDAL VOL I ml JJ *LJ jjljj 2J i 200 I;1i zvu BP mm H-g IooL T t 5 Aug o0 Ie sec 10,ug % Co2 RESP FLOW L /sec ij-7 I' j7 0 il I.\J i-i, i, V fi, 600 _ TIDAL 400 V VOL aou4i~iuis J4JUW mm M F" No T-T!I w. 200I IOOL t Augt 20,ug t 20 p FIG. 1.-Reflex effects of dopamine upon respiration and circulation of a dog. Upper panels and lower left: effects of 5, 10, and 20,pg injected into the left common carotid artery; all major branches ligated except the occipital artery. Lower right: effect of dopanine injected into the same artery after denervation of the left carotid body. From above downward: % C02 ill inspired and expired air; rate of inspiratory and expiratory air flow; respiratory tidal volume; femoral arterial blood pressure. In this and following figures the first arrow of each pair indicates the beginning of the injection and the second arrow indicates the end of the wash.

3 VOL. 59, 1968 PHYSIOLOGY: JACOBS AND COMROE 1189 cause they did not occur after the carotid nerve was blocked with procaine or cut, even though the occipital blood flow continued as before (Fig. 1). The minimal intracarotid dose of dopamine required to produce these effects was between 1 and 10,ug. When nicotine bitartrate, sodium cyanide, and dopamine were injected within a few minutes of each other, the ratios of their effective doses were approximately 1:10:10. Dopamine is, therefore, as effective as cyanide on a weight basis and about three times as effective as cyanide on a molar basis. To determine whether its known precursors have effects like those of dopamine, we injected ityrosine and -DOPA into the carotid catheter. As much as 10 mg of -tyrosine or 1 mg of -DOPA had no effect; 10 mg of L-DOPA produced no immediate response, although in four experiments on two dogs it did produce intense hyperpnea seconds after injection (Fig. 2). RESP 9 I FLOW 6F L /sec 3 J 600 r TIDAL ~~ VOL 400 m 2 00 J J JJ JJJJ IT I SJ 200 B P 100_ l mrn Hg J ij J J i J 10 mg /0 FIG. 2.-Effects of 10 mg DOPA injected into the right common carotid artery of a dog. Note that an increase in breathing does not occur until 30 sec after the beginning of the injection and intense hyperpnea does not occur until 40 sec after the beginning of the injection. Records from above downward: same as in Fig. 1. Since dopamine is converted either to norepinephrine or to homovanillic acid, we tested these two compounds for their effectiveness as carotid-body stimulants. Homovanillic acid, 10,gg-1 mg, had no effect in one dog; in a second dog, 1 mg elicited slight hyperpnea and 8.5 mg caused definite hyperpnea and slight hypotension. Norepinephrine produced hyperpnea of carotid-body origin in seven of ten dogs tested (Fig. 3); in the seven, the minimal effective intracarotid dose was between 10 and 40,gg; in the other three, the highest dose injected, 40 Atg, produced no hyperpnea. When dopamine and norepinephrine were injected within a few minutes of each other into the carotid catheter of the same dog, the ratio of the effective doses was approximately 1:5; in only one dog was the carotid body more sensitive to norepinephrine than to dopamine. We tested the effects of several blocking agents on the response to dopamine.

4 1190 PHYSIOLOGY: JACOBS AND COMROE PRoc. N. A. S. % C02 2 K Y1W l{v\ftnj47\ 0-J RZESP.6 FLOW.3II FIG. 3.-Effect of 100 pagof nor- L /sec 0 fjww epinephrine injected into the left common carotid artery of a dog. Records from above downward: TIDAL same as in Fig. 1. All major VOL 500 branches of the carotid artery were l 0 uiw~li2 Jr ijjjji w j ligated except the occipital artery. 200 n 3P 00 mm H9 I0; _ o Mploog /O sec The first of these was hexamethonium, a ganglionic blocking agent. When it is given intravenously to dogs that cannot excrete it in the urine (renal arteries and veins ligated), the concentration of hexamethonium in the blood remains high enough to block the actions of previously effective doses of acetylcholine and nicotine on the carotid body.8 In three dogs so prepared, the response to nicotine usually decreased after mg of hexamethonium; the response to dopamine decreased in one, but was greater in the other two (Fig. 4). The respiratory response of the dogs to inhalation of 5% 0r95% N2 remained unchanged. We also tested the effect of drugs that block a- and,3-adrenergic receptors. An a-adrenergic receptor blocking agent, phentolamine, in amounts that blocked the pressor effect of norepinephrine given intravenously, reduced the effectiveness of intracarotid doses of dopamine, norepinephrine, and sodium cyanide in four of five dogs tested. Phentolamine did not alter the responsiveness of-the carotid body to nicotine. 4- % C c 11 TIDAL r- VOL 50- J HI 11 Gs1 ml 0 JJJ w.d22~~ih~jj~ 50- Sp 00 mmnf19 -L _ A- 0 /0 see l0/g BEFORE C - 6 AFTER C- 6 FIG. 4.-Effect of intracarotid injection of dopamine before (left) and after (right) the intravenous injection of 50 mg of hexamethonium. The hexamethonium was sufficient to block the response of the carotid body to previously effective doses of nicotine. Records from above downward: % CO2 in inspired and expired air; respiratory tidal volume; femoral arterial blood pressure.

5 VOL. 59, 1968 PHYSIOLOGY: JACOBS AND COMROE A f3-adrenergic receptor blocking agent, propranolol, given in doses sufficient to block the effects of intravenous injections of isoproterenol, never decreased the effect of dopamine on the carotid body; in two of the three dogs tested, dopamine had considerably greater effect when given after propranolol. Because some chemical substances that stimulate the carotid bodies (e.g., nicotine) also excite the thoracic chemoreflex and produce reflex bradyeardia, hypotension, and apnea,9 we injected dopamine into the right atrium or ventricle in three dogs. As much as 1 mg of dopamine did not produce effects typical of the thoracic chemoreflex, although 100 jig of nicotine bitartrate was effective in the same animal. Dopamine in doses of ,ug raised systemic arterial blood pressure without causing the hyperpnea typical of carotid-body stimulation; larger amounts, 500 lg-1 mg, were required to cause such hyperpnea. In this respect dopamine again differs from nicotine; the first effect of nicotine, given intravenously in very low doses, is hyperpnea due to carotid-body reflexes. 10 Dopamine, injected into the common carotid artery after denervation of the carotid body and sinus, had no immediate effects that might be attributed to activation or depression of parts of the central nervous system, even though occipital blood flow into the circle of Willis continued as before. Discussion.-MIany chemical substances stimulate the carotid chemoreceptors. The finding that still another, dopamine, excites these cells is noteworthy because dopamine is believed to be a humoral transmitter in parts of the brain and is a normal constituent of carotid-body Type I cells. This raises the question whether dopamine plays a physiological role in excitation of the carotid body. It may do so indirectly by reducing blood flow through the carotid body without decreasing its metabolic rate, thus producing local hypoxia and accumulation of metabolically formed CO2 and hydrogen ions. Dopamine is reported to constrict some vascular beds in the dog."' 12 We have not attempted to measure blood flow through the carotid body because of technical difficulties, but decrease or cessation of blood flow through the carotid body should result in a gradual increase-in tidal volume and rate of ventilation.'3 14 The response to dopamine, however, was most intense with the first breath after injection. This leads us to believe that dopamine acts directly on sensory receptors to stimulate them. A direct action could be either pharmacological (like that of nicotine) or physiological. It is certain that dopamine does not act on those components of the carotid body that are stimulated by nicotine. Hexamethonium, in doses that reduced or blocked the response of the carotid body to nicotine, usually increased rather than decreased the hyperpnea caused by dopamine. The potentiation of the dopamine effect by hexamethonium could be due to decreased arterial blood pressure and decreased blood flow through the carotid body, which would increase the blood concentration of intraarterially injected dopamine or produce ischemia of the carotid body, or both. Large doses of hexamethonium might also cause respiratory depression and arterial hypoxemia which could sensitize the carotid chemoreceptors to the action of drugs. Catecholamines might act physiologically by stimulating a-receptors, f- receptors, or postulated dopamine receptors.'2 Large doses of propranolol

6 119'S PHYSIOLOGY: JACOBS AND COMROE PROC. N. A. S. failed to block the response to dopamine; indeed, the response to dopamine usually increased after propranolol. The effects of phentolamine were not consistent, although previous administration of phentolamine usually reduced the effectiveness of dopamine (and of sodium cyanide). Neither propranolol nor phentolamine blocked the hyperpnea or hypertension associated with systemic hypoxia. Since there is no specific antagonist of the postulated dopamine receptors, we were unable to determine whether block of these diminished carotid-body responses to hypoxia. We cannot determine by our experiments whether dopamine plays any direct or indirect physiological role in the activation of the carotid body. The failure in some experiments of intraarterial dopamine to stimulate the carotid body after phentolamine at a time when systemic hypoxia caused both hyperpnea and hypertension does not rule out the possibility that hypoxia may release dopamine from Type I cells in intimate relationship to sensory nerve endings that are unaffected by phentolamine given intravenously. We have not yet been able to deplete the (carotid body of catecholamines (by using reserpine, prenylamine, or similar agents) and thus determine whether this interferes with its chemosensory function. Although the carotid bodies of cats contain dopamine,6 7preliminary experiments in our laboratory have shown that dopamine does not excite their carotid, bodies although hypoxia, cyanide, and nicotine do. This might be evidence that dopamine is not an essential neurohumor for physiological activation of chemoreceptors in the cat or dog, since it seems unlikely that the basic mechanism for activating such a fundamental defense mechanism should differ in these two species. It is possible, however, that cats have a barrier to the transfer of dopamine from the arterial blood to the carotid body and dogs do not; such a barrier exists in many parts of the brain in cats and dogs. Biscoe and Stehbens" and Blumcke and associates"6 have recently suggested that the Type I cells, which contain most of the catecholamines in the carotid body, are not part of the chemosensory mechanism, but, instead, are similar to adrenal medullary cells that release catecholamines into the blood during severe hypoxia or asphyxia. If this is so, the action of dopamine and norepinephrine on the carotid-body sensory mechanism may be purely pharmacological. Conclusion.-Dopamine is a potent stimulant of the carotid bodies in the dog, and presumably acts directly on the sensory receptors. Its stimulant action is not prevented by prior administration of either a ganglionic blocking agent (hexamethonium) or a #3-adrenergic receptor blocking agent (propranolol). Further evidence is needed to assign a physiological role to dopamine in the excitation of these sensory receptors. * Supported by grants from the Office of Naval Research and the USPHS (HE-06285). t R. Byck, working in our laboratory, reported the relative effects on the carotid body of phenethylamine and 15 of its derivatives; 3-hydroxytyramine or dihydroxyphenethylamine (now called dopamine) was one of these (Federation Proc., 16, 287 (1957), abstract). ' Eyzaguirre, C., H. Koyano, and J. R. Taylor, J. Physiol., (London), 178, 463 (1965). 2 Muscholl, E., K.-H. Rahn, and M. Watzka, Naturwisenschaften, 47, 325 (1960). 3 Niemi, M., and K. Ojala, Nature, 203, 539 (1964). 4Pryse-Davies, J., I. M. P. Dawson, and G. Westbury, Cancer, 17, 185 (1964). 6 Hamberger, B., M. Ritzen, and J. Wersall, J. Pharmacol. Exptl. Therap., 152, 197 (1966).

7 VOL. 59, 1968 PHYSIOLOGY: JACOBS AND COMROE Fillenz, M., and R. I. Woods, J. Physiol., (London), 186, 39P (1966). 7 Chiocchio, S. R., A. M. Biscardi, and J. H. Tramezzani, Science, 158, 790 (1967). 8 Byck, R., Brit. J. Pharmacol., 16, 15 (1961). 9 Dawes, G., and J. H. Comroe, Jr., Physiol. Rev., 34, 167 (1954). 10 Comroe, J. H., Jr., and J. Nadel, in Tobacco and Health (Springfield, Ill.: Charles C Thomas, 1962), p "Eble, J. N., J. Pharmacol. Exptl. Therap., 145, 64 (1964). 12 McNay, J. L., R. H. McDonald, Jr., and L. I. Goldberg, Circulation Res., 16, 510 (1965). 13 Landgren, S., and E. Neil, Acta Physiol. Scand., 23, 158 (1951). 14 Lee, K. D., R. A. Mayou, and R. W. Torrance, Quart. J. Exptl. Physiol., 49, 171 (1964). 15 Biscoe, T. J., and W. E. Stehbens, Quart. J. Exptl. Physiol., 52, 31 (1967). 16 Blumcke, S., J. Rode, and H. R. Niedorf, Z. Zellforsch., 80, 52 (1967).