Carotid body chemoreceptors as a pharmacological model

167

TIPS - April 1985 transferrin has only a mildly acidic pH (6.5)24. In hepatocytes, internalized asialoglycoprotein has been localized by electron microscopy in endocytic vesicles and lysosomes, while its receptor appeared uniquely concentrated in tubular extensions of these endocytic vesicles and in an extended tubular network in the Golgi system. The receptor-rich, tubular network (CURL) was proposed as an intermediate in receptor recycling 7. An elegant double-label study was subsequently carried out, comparing distributions of receptors with different intracellular fates 21. The phosphomannosyl receptor and asialoglycoprotein receptors (which recycle, but whose ligands are largely lysosometargeted), were compared to the immunoglobin receptor (whose ligands, polymeric IgA in this case, are directed along with the receptor by 'transcytosis' to bile cannicular domains of the plasma membrane). All receptors were localized to a system of peripheral Golgi-associated tubules (CURL) 7, but a striking microheterogeneity was observed between the locations of the immunoglobin receptor and the other two. Dynamic, time course electron microscope studies in cultured cells 11,12 have been able to directly visualize similar CURL vesicles and tubules involved in transferrin-receptor recycling. These data suggest that CURL participates in sorting of receptors and ligands destined for various plasma membrane and lysosomal destinations. It is not yet known whether recycled receptors converge with other newly biosynthesized, secreted proteins in a common secretory pathway. However, the whole system is obviously reminiscent of Farquhar's original work on commonality of the secretory pathways for biosynthesized immunoglobins and endocytosed membrane tracers in cultured myeloma cells 1. Future studies will probably continue to utilize monoclonal antibody technology to attempt to define biochemical routing tags of receptors, and to establish the enzymatic and structural character of the various endocytic subcompartments. In the meantime pharmacologists may learn to take advantage of the endocytic route of attack of cells in

design of better chemotherapeutic agents and in elucidation of new mechanisms and sites of drug resistance.

12 Willingham, M. C., Hanover, J.A., Dickson, R. B. and Pastan, I. (1984) Proc. Natl Acad. USA 81, 175-179 13 Klausner, R. D., van Renswoude, J., Kempf, C., Rao, K., Bateman, J. L. and Robbins, A.R. (1984) J. Cell Biol. 98,

References

14 Goldfine, I. D. (1981) Biochem. Biophys. Acta. 650, 53-67 15 Novikoff, A. B. and Novikoff, P.M. (1977) Histochem. J. 9, 525--552 16 Hanover, J. A., Willingham, M. C. W. and Pastan, I. (1984) Cell 39, 283-293 17 Krupp, M. N., Connolly, D.T. and Lane, M.D. (1982) J. Biol. Chem. 257, 11489-11496 18 Beguinot, L., Lyan, R. M., Willingham, M.C. and Pastan, I. (1984) Proc. Natl Acad. Sci. USA 81, 2384-2388 19 Neufeld, E. F. and Ashwell, G. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, W. J., ed), pp. 241-266, Plenum Press, New York 20 Brown, W. J. and Farquhar, M. G. (1984) Cell 36, 295-307 21 Geuze, H. J., Slot, I.W., Strous, G. J. A. M., Peppard, J., yon Figura, K., Hasilik, A. and Schwartz (1984) Cell 37, 195-204 22 WiUingham, M. C., Pastan, I.J. and Sahagian, G.G. (1983) J. Histochem. Cytochem. 31, 1-11 23 Sahagian, G. G. and Neufeld, E. (1983) J. Biol. Chem. 258, 7121-7128 24 Yamashiro, D. J., Tycko, B., Fluss, S. R. and Maxfield, F. R. (1984) Cell 37, 789-

1098-1101

1 Farquhar, M. G. and Palade, G. E. (1981) J. Cell. Biol. 91, 775-1035 2 Steinman, R. M., Mellman, I. S., Muller, W. A. and Cohn, Z. A. (1983) J. Cell Biol. 91, 775-1035 3 Brown, M. S., Anderson, R. G. W. and Goldstein, J. L. (1983) Cell 32, 663--667 4 King, A. C. and Cuatrecasas, P. (1981) N. Engl. J. Med. 305, 77M38 5 Pastan, 1. and Willingham, M. C. (1983) Trends Biochem. Sci. 8, 245-249 6 Helenius, A., Mellman, I., Wall, D. and Hubbard, A. (1983) Trends Biochem. Sci. 8, 250-254 7 Geuze, H. J., Slot, J.W., Strous, G. J. A. M., Lodish, H. F. and Schwartz, A. L. (1983) Cell 32, 277-287 8 Klausner, R. D., Van Reswoude, J., Ashwell, G., Kempf, C., Schecter, A. N., Dean, A. and Bridges, K.R. (1983) J. Biol. Chem. 254, 8847-8854 9 Dickson, R. B., Beguinot, L., Hanover, J. A., Richert, N. D., Willingham, M. C. and Pastan, I. (1983) Proc. Natl Acad. Sci. USA 80, 5335-5339 10 Dickson, R. B., Hanover, J. A., Willingham, M. C. and Pastan, I. (1983) Biochemistry 22, 5667-5674 11 Hopkins, C. R. (1983) Cell 35, 321-330

80O

Carotid body chemoreceptors as a pharmacological model Daniel S. McQueen Carotid chemoreceptors provide a model for studying the actions of peptides and other substances in an isolated organ under controlled conditions using a variety of techniques, e.g. neuropharmacological, radioligand binding, immunocytochemical, biochemical and biophysical. It should be possible, for example, to determine whether chemodepression evoked by enkephalins results from changes in Ca 2+ uptake, and~or in cAMP levels, which in turn cause a reduction in transmitter release. As far as the physiological role of the enkephalins or other peptides is concerned, it will be possible to measure their release from the carotid body in response to physiological and pharmacological stimuli in vitro. Daniel S. McQueen suggests that there are plenty of exciting prospects for using the carotid body as a model to investigate the pharmacology of polypeptides and their interactions with other substances. For nearly 60 years it has been known that the carotid body, a small organ (approx. 1 mm 3) situated in the carotid bifurcation (Fig. la), can evoke various reflexes, the best known of which is hyperventilation in response to

Daniel S. McQueen is a Reader in the Department of Pharmacology, University of Edinburgh Medical School, Edinburgh EH8 9JZ, UK.

hypoxia. The sensors in the carotid body are called arterial chemoreceptors and much work has gone into trying to discover how they function, for example by investigating the mechanisms involved in monitoring arterial blood gas tensions/pH and in controlling sensory nerve discharge. However, despite a variety of hypotheses, no generally accepted account of the process has yet emerged 1-s.

1985,ElsevierSciencePublishersB.V.,Amsterdam 0165- 6147/85/$02.00

TIPS - April 1985

168 Early experiments on carotid chemoreceptors led to the suggestion that, b y analogy with motor nerves, chemical transmission might be involved in chemoreception: type I cells m a y release a transmitter in response to hypoxia or hypercapnia, t h e r e b y activating 'post-synaptic' sensory nerves (Fig. lb). Since then, various putative transmitters in other parts of the nervous system have been found in the carotid b o d y 3,4, including: noradrenaline, adrenaline, d o p a m i n e (DA), 5-HT serotonin, acetylcholine (ACh), substance P, [Met]enkephalin, [Leu]enkephalin, vasoactive intestinal p o l y p e p t i d e (VIP), adenosine, ATP, glutamate and taurine. None of these has yet been shown, unequivocally, to be a chemosensory transmitter. This could m e a n that chemical transmission is not involved in the transduction process. However, other possibilities exist. One (or more) of these substances could be a chemosensory transmitter, b u t limitations in the experiments performed have not yet allowed this to be d e m o n strated. Alternatively the transmitter m a y be an u n k n o w n substance.

Methods of studying chemoreceptors Various techniques and species have been used to s t u d y carotid chemoreceptors 4. These include: reflex changes, e.g. respiratory or cardiovascular changes in whole animals, extracellular recording from sensory nerves in vivo, or in vitro (using excised carotid b o d y preparation), intracellular recording in vitro using carotid b o d y 'slices' in w h i c h cells and nerve endings can be visualized directly and substances a p p l i e d locally, electrophysiological studies on cultured chemoreceptor cells, receptor labelling using radioligands, biochemical measurements, i m m u n o c y t o c h e m i s t r y and histofluorescence, and gas chromatography-mass spectrometry. Some of the techniques are difficult because of the small sizes of the carotid b o d y and the type I cells (10 ~m), b u t techniques are improving. It is particularly advantageous that animals have two carotid b o d i e s because one of the pair can be used for pharmacological studies while the other serves as a control, or is used for com-

p l e m e n t a r y experiments. Much of our k n o w l e d g e of chemoreceptor pharmacology has come from experiments in w h i c h drugs were used to p r o b e the chemosensors. Some advances will be illustrated b y briefly considering the p h a r m a c o l o g y of three substances present in the carotid b o d y , n a m e l y ACh, [Met]e n k e p h a l i n and d o p a m i n e .

Acetylcholine The discovery in the 1930s that A C h stimulates carotid chemoreceptors x,s led to proposals that it m i g h t be involved in chemoreception as an excitatory transmitter, a concept referred to as the 'cholinergic hypothesis'. Ganglion blocking drugs such as h e x a m e t h o n i u m and m e c a m y l a m i n e a n t a g o n i z e d chemoexcitatory actions of ACh and nicotine, whereas atropine d i d not 1,s. Quantitative neuropharmacological studies in vivo have s h o w n that although responses to exogenous ACh are reduced b y ganglion blocking drugs, and p o t e n t i a t e d b y anticholinesterases such as physostigmine, responses to physiological stimuli remain largely unaffected 6. This and other evidence, such as that, in rabbits, A C h causes c h e m o d e p r e s s i o n rather than excitation 4 countered the cholinergic hypothesis, b u t nonetheless i n d i cated that ACh receptors are present in the carotid b o d i e s of m a n y species. The presence of ACh in the carotid b o d y has been d e m o n strated b y gas c h r o m a t o g r a p h y mass spectroscopy 7 and, because ACh levels are not significantly affected b y chronic denervation of the carotid body, it is p r e s u m e d that ACh is located in cells, which are largely unaffected b y denervation, rather than in nerve terminals which degenerate after denervation 3. Type I and p o s s i b l y type II cells have a high affinity choline uptake m e c h a n i s m 5 which is unaffected b y denervation. Binding of radiolabelled c~-bungarotoxin has been s h o w n to occur at sites on type I ceils, but not to nerves in the carotid b o d y s. It has recently been p r o p o s e d that there are two classes of type I cell (type IA and type IB) based on the greater b i n d i n g affinity of type IB for 0¢-bungarotoxin. Type IA and type IB m a y represent functionally distinct cell types in the rat 9. If, as is often as-

sumed, 0~-bungarotoxin is a marker for the nicotinic ACh receptor, then chemoexcitation evoked b y ACh or nicotine appears to result from effects on cells rather than from direct actions on sensory nerve terminals 3. H o w ever, intracellular recordings in vitro have s h o w n that A C h has variable effects on the m e m b r a n e potentials of type I cells3. Neuropharmacological experiments on carotid b o d y slices 1° have revealed that nicotinic agonists depolarize type I cells, an action blocked b y 0c-bungarotoxin. Muscarinic agonists can also depolarize the cells; their action is blocked b y atropine b u t unaffected b y 0c-bungarotoxin. It is difficult to k n o w w h a t significance should b e attached to changes in the m e m b r a n e potentials of heterogenous type I cells in vitro in response to ACh and related drugs because in-vivo studies in cats indicate that m u s carinic agonists have little effect on chemosensory activity 11. 0cBungarotoxin is only a very w e a k antagonist of A C h - i n d u c e d chemoexcitation 6 and although c~bungarotoxin appears to be a ligand for nicotinic ACh receptors, it does not always block nicotinic actions. Recent evidence 12 suggests that K-bungarotoxin is a better p r o b e than ~bungarotoxin for neuronal receptors and m a y block b y acting at a site other than that recognized b y 0~-bungarotoxin. Changes in m e m b r a n e potential of type I cells m a y lead to the release of substances, such as ACh, DA a n d p e p tides w h i c h will in turn affect other cells and nerve terminals and m o d i f y chemosensory activity. Future pharmacological studies should p r o v i d e more information on ACh receptors and the role of ACh in chemoreception.

[Met]enkephalin The presence of p o l y p e p t i d e s in the carotid b o d y was predicted from the classification of type I cells as m e m b e r s of the a m i n e precursor uptake and decarboxylation(APUD) cell series 13. The cat carotid b o d y has s u b s e quently been s h o w n to contain at least four p e p t i d e s ([Leu] and [Met]enkephalin, substance P and VIP - or closely related i m m u n o reactive substances) 14. Both enkephalins are e q u i p o t e n t i n h i b i t o r s

169

TIPS - April 1985

of chemosensory discharge when administered close-arterial to the carotid body. This effect seems to involve opioid receptors since it can be antagonized by naloxone 4A5. Further characterization of the receptor(s) using selective antagonists is still needed, but the relatively low potency of naloxone coupled with the weak chemodepression evoked by morphine indicates that enkephalins may act on a b type receptor to depress discharge. This is supported by results from recent experiments which show that the selective opioid b receptor antagonist ICI 154, 129 (N,N-bisallyl-Tyr-Gly-Gly~-(CH2S)-Phe-Leu-OH)16 reduces [Met]enkephalin-induced chemodepression in cats (McQueen, unpublished observations). [Met]enkephalin-like material appears to co-exist with catecholamines in type I cells, possibly within the same vesicles 17, which raises the possibility that opioids may be coreleased with the amine(s), as they are in the adrenal medulla. Chemodepression evoked by [Met]enkephalin lasts longer than that caused by DA and is unaffected by the DA antagonist c¢-flupenthixol4; the response to DA is not significantly affected by naloxone. Neither ICI 154,129 nor naloxone has much effect on chemosensory discharge in anaesthetized cats breathing air, which suggests that

under these conditions chemoreceptors are not tonically inhibited by opioids. Dopamine Dopamine in the carotid body is released during hypoxiaTM. Generally, exogenous DA depresses chemosensory discharge recorded in vivo 4 and recent pharmacological studies using selective agonists (LY 141865, SKF 38393) and antagonists (domperidone, sulpiride) suggest that chemodepression results from actions at a DA D 2 receptor 19. The finding that DA does not increase cAMP levels in the carotid body 19 supports neuropharmacological evidence. (The D 2 receptor, unlike the D1, is not positively coupled to adenylate cyclase and so would not be expected to increase cAMP levels when activated.) If D2 receptors are indeed associated with different elements of the carotid body, it will be necessary to determine whether the action of DA on those receptors associated with nerves produces an effect on chemosensory discharge different from that evoked by actions on the cells. Experiments on rabbit carotid bodies showed that [3H]domperidone binds both to cells and nerves, and chronic denervation reduced binding by 30--60%. Treatment of rats with 6-OH DA has no significant effect on either

~ , toCNS

the DA content of the carotid body or on chemosensory responses to hypoxia s. There is evidence that DA can cause chemoexcitation, particularly in vitro 3, and DA generally depolarizes type I cells in vitro 3, although this effect is sometimes preceded by slight hyperpolarization 2°. It is not clear how changes in membrane potential affect chemosensory discharge. Pharmacological experiments have helped in understanding the role of DA in the carotid body. In addition they illustrate how suitable the carotid body is as a general model for studying drug actions. For example, the question of whether DA receptors become 'supersensitive' after chronic treatment with a DA antagonist (domperidone) was investigated using a combination of radioligand binding and neuropharmacological techniques in rabbits 21. There was a significant increase in the number of specific D2 binding sites in membranes prepared from the carotid bodies of chronically treated rabbits. Recordings of chemosensory discharge showed that this change in receptor density was accompanied by an increased sensitivity to the chemodepressant effects of DA. Thus the carotid body provided evidence that functional responsiveness altered and the number of D2

to CNS

~(petroual)

sensory cell body

sensory afferent motor afferent carotid sinus nerve

ganglion [ ~ glos ..s~,..,h,ar~ingeal (IX) nerve /~b ~ carotid sinus nerve ~ 2 ~ , ~ internal carotid artery

sy~pse

type I cell type II cell ~ a

"- external carotid artery common carotid artery

blood vessel b

sympathetic efferent

Fig. 1 Carotid body: (a) gross anatomy (b) simplified schematic representation of the receptor complex. Individual nerve fibres typically course to 10-20 type I cells. Not all type I calls receive a nerve supply and the surrounding glial-like type II cells do not appear to be innervated. The carotid sinus nerve contains both myellnated and unmyelinated sensory (afferent) fibres as well as motor (parasympathetic efferent) fibres. The presence of vesicles in type I cells suggests that chemical transmtssion may occur at the cleft or 'synapse' between the cell and the nerve-ending. Parasympathetic efferents could release transmitter(s) to affect the calls or nearby sensory terminals, providing efferent control of the chemoreceptors. This is in addition to that achieved by sympathetic nerves, which are able to influence sensory discharge by altedng blood flow through the organ. The question of whether the cells or the afferent nerve endings are the primary sensory element in the receptor complex has yet to be answered3-~

170 b i n d i n g sites i n c r e a s e d f o l l o w i n g c h r o n i c b u t n o t acute t r e a t m e n t w i t h d o m p e r i d o n e . It is n o t e s s e n tial to u s e c h e m o s e n s o r y disc h a r g e to m o n i t o r the i n t e r a c t i o n b e t w e e n DA, or o t h e r d r u g s , a n d r e c e p t o r s in the carotid b o d y . S i m i l a r i n f o r m a t i o n can be o b tained indirectly by recording v e n t i l a t i o n , e.g. in an a n a e s t h e t i z e d rat a n d b y i n j e c t i n g D A intravenously. The dose-related hypov e n t i l a t i o n that e n s u e s is c a u s e d m a i n l y b y the c h e m o d e p r e s s a n t action of D A on D 2 r e c e p t o r s in the carotid b o d y r e m o v i n g the p e r i p h e r a l d r i v e to b r e a t h i n g 22. N e o n a t a l t r e a t m e n t of rats w i t h capsaicin, w h i c h causes d e g e n e r a t i o n of u n m y e l i n a t e d p r i m a r y afferent n e u r o n e s , r e d u c e s t h e s e n s i t i v i t y of p e r i p h e r a l c h e m o r e c e p tors to h y p o x i a a n d i n c r e a s e s D A levels in the carotid b o d y 23. The levels of D A in t h e carotid b o d i e s of infants w h o s e d e a t h is a t t r i b u t e d to s u d d e n i n f a n t d e a t h s y n d r o m e (SIDS, cot death) are also s i g n i f i c a n t l y increased. T h e s e infants die f r o m a p n o e a d u r i n g sleep 24 a n d are t h e r e f o r e p r e s u m e d n o t to h a v e n o r m a l c h e m o r e f l e x v e n t i l a t o r y a n d arousal r e s p o n s e s to h y p o x i a . It s e e m s p o s s i b l e , then, that rats treated n e o n a t a l l y w i t h c a p s c a i c i n c o u l d p r o v i d e an a n i m a l m o d e l for SIDS.

TIPS - A p r i l 1985

A l t h o u g h m u c h r e m a i n s to b e learned about the pharmacology of carotid c h e m o r e c e p t o r s , the information already available s h o w s that the carotid b o d y can s e r v e as a g e n e r a l m o d e l for s t u d y i n g d r u g actions (e.g. D A o n D2 receptors) a n d i n t e r a c t i o n s ( a m i n e s / p e p t i d e s ) o n a v a r i e t y of receptors. T h e carotid b o d y is small, b u t is r e a d i l y i s o l a t e d a n d can b e s t u d i e d in vitro as w e l l as in vivo; controlled s t i m u l i can easily be a p p l i e d to t h e s e n s o r y r e c e p tors, a n d the o u t p u t f r o m the r e c e p t o r c o m p l e x can be m e a s u r e d quantitatively. References

1 Anichkov, S. V. and Belen'kii, M.L. (1963) Pharmacology of the Carotid Body Chemoreceptors, Pergamon, Oxford 2 Biscoe, T. J. (1971) Physiol. Rev. 51, 437495 3 Eyzaguirre, C. and Fidone, S. (1980) Am. J. Physiol. 239, C135-152 4 McQueen, D. S. (1983) in Physiology of the peripheral arterial chemoreceptors

(Acker, H. and O'Regan, R. G., eds), pp. 149-195, Elsevier, Amsterdam 5 Eyzaguirre, C., Fitzgerald, R. S., Lahiri, S. and Zapata, P. (1983) in Handbook of Physiology. The Cardiovascular System III, Peripheral Circulation Part II

(Shepherd, J.T. and Abboud, F.M., eds), pp. 557-621, Am. Physiol. Soc. 6 McQueen, D. S. (1977) J. Physiol. 273, 515-532 7 Fidone, S. J., Weintraub, S. and Stavinoha, W.B. (1976) J. Neurochem.

26, 1047-1049 8 Dinger, B., Gonzalez, C., Yoshizaki, K. and Fidone, S. (1981) Brain Res. 205, 187-193 9 Chen, L-L. and Yates, R. D. (1984) J. Neurocytol. 13, 281-302 10 Eyzaguirre, C. and Monti-Bloch, L. (1982) Brain Res. 252, 181-184 11 McQueen, D. S. (1978) Q. J. Exp. Physiol. 63, 171-178 12 Chiappinelli, V. A. (1984) Trends Pharmacol. Sci. 5, 425-428 13 Pearse, A. G. E. (1969) J. Histochem. Cytochem. 17, 303-313 14 Wharton, J., Polak, J.M., Pearse, A. G.E., McGregor, G.P., Bryant, M.G., Bloom, S.R., Emson, P.C., Bisgard, G.E. and Will, G.A. (1980) Nature (London) 284, 269-271 15 McQueen, D. S. (1983) Br. Med. Bull. 39, 77-82 16 Shaw, J. S., Miller, L., Turnbull, M.J., Gormley, J.J. and Morley, J.S. (1982) Life Sci. 31, 1259-1262 17 Hansen, J. T., Brockaw, J., Christie, D. and Karasek, M. (1982) Anat. Rec. 203, 405-410 18 Gonzalez, C. and Fidone, S. (1977) Neurosci. Left. 6, 95-99 19 Mir, A. K., McQueen, D. S., Pallor, D. J. and Nahorski, S.R. (1984) Brain Res. 291, 273-283 20 Matsumoto, S., Nakajima, T., Uchida, T., Ozawa, H. and Ushiyama, J. (1982) Brain Res. 239, 674-678 21 McQueen, D. S., Mir, A.K., Brash, H. M. and Nahorski, S. R. (1984) Eur. J. Pharmacol. 104, 39-46 22 Cardenas, H. and Zapata, P. (1981) Neurosci. Lett. 24, 29-33 23 McQueen, D. S. and Mir, A. K. (1984) Br. J. Pharmacol. 83, 909-918 24 Perrin, D. G., Cutz, E., Becker, L.E., Bryan, A. C., Madapallimatum, A. and Sole, M. J. (1984) Lancet ii, 535-537