Trp7' 9 lo-sp. adrenoceptor antagonists in order to establish the nature of neurotransmitters

Transcription

1 Journal of Physiology (199), 43, pp With 5 figures Printed in Great Britain ALTERATIONS IN CAT KNEE JOINT BLOOD FLOW INDUCED BY ELECTRICAL STIMULATION OF ARTICULAR AFFERENTS AND EFFERENTS BY A. KHOSHBATEN AND W. R. FERRELL From the Institute of Physiology, University of Glasgow, G12 8QQ (Received 1 January 199) SUMMARY 1. Experiments were performed in cats anaesthetized with pentobarbitone. Laser Doppler flowmetry was used to assess the responses of knee joint blood vessels to nerve stimulation under control conditions and in the presence of different adrenoceptor antagonists in order to establish the nature of neurotransmitters released from articular nerve fibres. 2. The posterior articular nerve (PAN) supplying the knee was stimulated at different intensities, and frequency-response curves were obtained. In fourteen animals electrical stimulation of PAN produced an initial vasoconstriction during stimulation which in eight of these was followed by a prolonged dilatation on cessation of stimulation. The constrictor response was increased as a function of frequency but was little altered with increasing intensity beyond a threshold level. 3. The constrictor response to electrical stimulation of PAN was markedly reduced by the a-adrenergic antagonist phentolamine (1-5 M), the al-blocker prazosin (1-5 M), and guanethidine (1-5 M) which inhibits the release of noradrenaline, ATP, and neuropeptide Y from sympathetic nerve endings. 4. The constrictor response to PAN stimulation was unaffected by the ot2-blocker rauwolscine and the P2-purinoceptor desensitizer a,,/-methylene ATP. 5. The dilator response was due to activation of afferent fibres as it could also be produced by direct electrical stimulation of the L7 dorsal roots. 6. The dilator response to stimulation of PAN or the L7 dorsal root was reduced by prior intra-articular injection of 1 jug of the substance P antagonist D-Pro4-D- Trp7' 9 lo-sp 7. These results suggest that the vasoconstrictor response to electrical stimulation of PAN is most likely to be mediated via noradrenaline acting mainly upon ocladrenoceptors. As the dilator response to articular nerve stimulation is reduced by a substance P antagonist, the mediator inducing this response may be substance P or a related neurokinin. INTRODUCTION Although there have been some studies concerning the innervation of the knee joint in the cat (Gardner, 1944; Freeman & Wyke, 1967), less is known about the action of joint nerves in the regulation of articular blood flow. Cobbold & Lewis MS 8191

2 78 A. KHOSHBATEN AND W. R. FERRELL (1956) observed that electrical stimulation of the medial articular nerve (MAN) resulted in reduction of blood flow to the dog knee joint. However, the electrical stimulus parameters used were not stated. In a more recent investigation, electrical stimulation of the posterior articular nerve (PAN) was found to produce an initial vasoconstriction of blood vessels in the cat knee (Ferrell & Cant, 1987). However, in neither of these investigations was the nature of the neurotransmitters mediating these responses examined. In the study of Ferrell & Cant (1987) it was also observed that neurogenically mediated vasodilatation occurred after PAN stimulation. These authors concluded that stimulation of unmyelinated efferent nerve fibres, which are known to be present in PAN (Langford & Schmidt, 1983), produced the initial vasoconstriction whilst the vasodilatation resulted from activation of unmyelinated afferents. These findings were confirmed in a more recent study in the rabbit by Khoshbaten & Ferrell (199). It was shown that stimulation of nerves supplying knee joint blood vessels in the rabbit resulted in vasoconstriction during stimulation followed by a prolonged vasodilatation on cessation of stimulation. In other sites such as skin, it has been observed that a neurogenically mediated increase in blood vessel permeability is accompanied by dilatation of these blood vessels (Couture & Cuello, 1984). For many years, studies of the neurohumoral control of the vasculature have been dominated by the role of catecholamines released from sympathetic perivascular nerves and from the adrenal medulla into the blood stream (Burnstock, 1975; Burnstock, Chanley & Campbell, 1986). Attention has also been paid to the cholinergic innervation of some blood vessels (Burnstock, 198). In the last decade, however, non-adrenergic, non-cholinergic components of the autonomic nervous system have become established. Many neurotransmitters such as ATP, vasoactive intestinal polypeptide, substance P, dopamine and neuropeptide Y are now considered to play a role in the regulation of blood vessel calibre in many vascular beds (Burnstock, 1985). However, whether any of these substances are released from nerves innervating articular blood vessels has not been established. There is evidence for the involvement of noradrenaline as a neurotransmitter in nerves supplying rabbit knee joint blood vessels. In an in vitro study carried out by Ferrell & Khoshbaten (199) it was found that the vasoconstrictor response to electrical stimulation of the rabbit knee joint capsule was mediated via noradrenaline acting upon ax-adrenoceptors. The dilator response observed by Ferrell & Cant (1987) was abolished by intra-articular injection of a substance P antagonist, suggesting that this was mediated by substance P or a related neuropeptide. The present study was performed to further examine the response of cat knee joint blood vessels, as measured by laser Doppler flowmetry, to electrical stimulation of the nerve supply to the posterior aspect of the knee, and determine the nature of neurotransmitters released from nerve endings on these vessels. METHODS Experiments were performed on fourteen adult cats, their weights varying between 1-8 and 4.5 kg. Anaesthesia was by intraperitoneal injection of pentobarbitone (Sagatal, May & Baker Ltd) in a dose of 45 mg kg-'. Maintenance of anaesthesia was by means of further intra-arterial injections of pentobarbitone in doses of 3-1 ml (3 mg ml-'), depending on the weight of cat. The

3 NEURAL REGULATION OF KNEE JOINT BLOOD FLOW right common carotid artery was exposed and cannulated for monitoring arterial blood pressure. A laminectomy was performed to expose the spinal cord, and isolation of the dorsal roots L7 and S1. After laminectomy PAN was also prepared for electrical stimulation. Relative changes in knee joint blood flow were assessed by laser Doppler flowmetry (Moor Instruments MBF 3). The fibre- A Capsule 79 T'ibia emur r Nee Pate a Probe C L5 Ei 3 : 2 o1 en) 1 1 min Fig. 1. Diagrammatic representation of (A) the lateral aspect of the cat knee joint and (B) the posterior aspect showing the intra-articular location of the laser probe which was inserted from the antero-lateral side through the infra-patellar region. C, responses to stimulation of PAN (1 V, 1 Hz, 1 ms) with the laser probe sited externally on the surface of the postero-medial capsule. The arrow indicates that a black polythene film (opaque to laser radiation) located between the internal surface of the posterior capsule and the medial femoral condyle was removed. Bars indicate the period of stimulation. Little change occurred in either the basal flowmeter signal or the response to nerve stimulation. optic probe (2 mm in diameter) was either located on the external surface of the posterior capsule or a smaller probe ( 9 mm in diameter) was inserted (via a 18 gauge needle) from the antero-lateral side of knee into the joint cavity and advanced dorsally to contact the internal surface of the posterior capsule (Fig. 1A and B). These probes contained two fibres, one of which conducted laser radiation (78 nm) to the tissue with the other fibre conducting the back-scattered light to

4 8 A. KHOSHBA TEN AND W. R. FERRELL photodetectors. The rectal temperature was maintained at 37+1 C by a heating lamp. The temperatures of the spinal and popliteal paraffin pools were also maintained at the same level by means of additional heating lamps. Stimulation of L7 and Si In experiments involving electrical stimulation of the L7 and SI dorsal roots, these were sectioned close to the spinal cord and placed on a pair of silver electrodes with an inter-electrode distance of 2-5 mm. These electrodes were connected to a Harvard advanced stimulator which produced squarewave pulses whose duration, intensity, and frequency could be varied. The parameters used for both PAN and dorsal root stimulation were chosen to be suprathreshold for group IV afferent fibres, using the criteria of Sato, Schaible & Schmidt (1983), and are included in the figure legends. Stimulation of PAN In experiments where PAN was to be electrically stimulated, it was necessary to transect this nerve to avoid reflex alterations in systemic blood pressure. Prior to transection, PAN was located on a pair of silver electrodes and then cut proximally whilst knee joint blood flow, arterial blood pressure, and heart rate were monitored continuously on a pen recorder. Although only shown once in subsequent figures, blood pressure and heart rate were monitored throughout the experiments and recordings only obtained whilst these remained constant. Intra-articular placement of the flowmeter probe resulted in only the capsular tissue being sampled, although this did not distinguish between synovial and peri-articular blood flow. Placing the probe on the external aspect of the posterior capsule could potentially sample structures other than the capsule (e.g. ligaments, bone). To test this, control experiments were performed which indicated that the flowmeter signal only related to changes in blood flow in the posterior region of the capsule (Fig. 1C). Drug administration Adrenoceptor antagonists were administred by bolus injection (-2 ml) of these agents via a polythene cannula inserted into an artery distal to the popliteal artery. The cannula was then advanced proximally until the tip was placed just distal to the knee joint supply and tied into position. The substance P antagonist D-Pro4-D-Trp7 9,'-SP4.11 was administered by intra-articular injection in a dose of 1 jug and a volume of -5 ml. Control experiments indicated that a similar volume of saline had little effect on nerve-mediated responses. Statistical data analysis was carried out by either paired or unpaired t test. An F test was also used to test the assumption of homogeneity of variances. Where this exceeded tabled F values, modified t values were generated using the formula described by Phillips (1978). All data expressed on graphs are means + S.E.M. Differences between means were considered significant if the P values were 5 % or less. RESULTS Transection of PAN As shown in Fig. 2, in some experiments it was observed that transection of PAN resulted in an obvious rise in blood flow to the joint which was not accompanied by any reflex rise in arterial blood pressure, suggesting loss of pre-existing sympathetically mediated vasoconstrictor 'tone'. Electrical stimulation of PAN In all fourteen cats a constrictor response was observed on electrical stimulation of PAN (Fig. 3A). Responses to PAN stimulation were obtained both by increasing the number of pulses per second and also by increasing the stimulus voltage (Fig. 4). These showed considerable enhancement of constrictor response to increasing the frequency (with a maximum at 3 Hz) but little significant change to increasing the voltage. As illustrated in Fig. 3A there was a consistent response to PAN stimulation

5 NEURAL REGULATION OF KNEE JOINT BLOOD FLOW at intervals of 5 min. In eight out of fourteen cats, stimulation of articular nerve fibres resulted in a characteristic pattern of a rapid initial fall in the blood flow of the knee joint during stimulation followed by a prolonged vasodilatation on cessation of stimulation (Fig. 5A). It was noticeable that in these animals the >4-, 3-._ <o 2 4- E 1- LL -r *r _ 81 1 min I CD E E a c E CA) Ir li Fig. 2. Acute transection of PAN (at the arrow) induces a rise in knee joint blood flow with little change in blood pressure (BP) or heart rate (HR), presumably due to removal of sympathetic vasoconstrictor 'tone'. constrictor response was rapid in onset, in many cases reaching maximum within - 15 s of the onset of the stimulus pulse train (Fig. 5A). In those animals not displaying a dilator response, the constrictor response was much slower in onset, often reaching maximum after the end of the stimulus pulse train, and taking several minutes to recover (Fig. 3A). These different patterns of constrictor response to nerve stimulation were always consistent in a given animal. The effect of different antagonists on PAN stimulation The object of these experiments was to assess whether PAN stimulation mediates its constrictor effect via noradrenaline or other mediator(s) released from sympathetic efferent fibres. As shown in Fig. 3B, close intra-arterial injection of either

6 82 A. KHOSHBA TEN AND W. R. FERRELL A 3~- E X- 2 \ 1 1m ou_.,n _,_.,. 1 min SAL RAW a, f-matp Phentolamine B -o. -. o 525 ct4 5 *** *** ** n.s. n.s., GUA PHA PRA RAW (x, /3-mATP Fig. 3. A, PAN stimulation (1 Hz, 1 V, 1 ms) induces vasoconstriction in articular blood vessels which was unaffected by -2 ml 9 % saline (SAL), rauwolscine (RAW 1-s M), or a, /J-mATP (1-s M), which were administered intra-arterially prior to nerve stimulation. The vasoconstrictor response was not only abolished by administration of phentolamine (1-s M), but a dilator response also became evident. The filled bars denote periods of PAN stimulation. B, the constrictor response to PAN stimulation is shown before (filled histogram) and after administration of different antagonists. GUA, guanethidine (t1- M); PHA, phentolamine (1-' M); PRA, prazosin (t- M); RAW, rauwolscine (1-' M); and a,,/-matp (1-s M). n = 5-8. n.s. = not significant; **P < -1; ***P < ', g 5/ ~ =_ 9~~~~~ I I Frequency (pulses s-1) Fig. 4. Constrictor response as a function of frequency of PAN stimulation at 5 V (@), and 1 V () pulse amplitudes (width 1 ms). No significant changes are observed between the two voltages. n = 5-12.

7 NEURAL REGULATION OF KNEE JOINT BLOOD FLOW 83 A S 5- -E 4' CD ) +,, a) 2- E 3 1 L -~~~wakw*t* ii 1 min B 71i 4@, X 4- c 3. o.2 2- a 1 min a~~~~~~~~~~~~~~~~~~ f 1 b 1 min c * * 2 4-c -_ou37s.5[[. L7S1 L7 SPA L7 P+B P+B PAN Fig. 5. A, changes in the flowmeter signal in response to electrical stimulation of PAN (1 V, 1 Hz, 1 ms). In this animal, vasoconstriction occurs rapidly during stimulation reaching its maximum within 15 s of onset, followed by dilatation on cessation of stimulation. Filled bars denote periods of stimulation. B, traces showing articular blood flow changes when the L7 dorsal root was stimulated (1 Hz, 1 V, 1 ms) before (a) and after (b) intra-articular injection of l1 g of the substance P antagonist D-Pro4-D- Trp7 9' l-sp 411 which significantly attenuates the dilator response. Filled bars indicate period of stimulation. Flowmeter time constant 1 s in a and 3 s in b. C, comparison of the dilator response to electrical stimulation of L7 versus S1; L7 stimulation before and after intra-articular injection of D-Pro4-D-Trp7 9' lo-sp4-11 (SPA); L7 stimulation compared to PAN stimulation after intra-arterial injection of the a blocker phentolamine (P + B); and PAN stimulation before and after phentolamine (P + B). Response to L7 stimulation was significantly reduced by SPA. L7 stimulation was more effective in eliciting a dilator response than PAN stimulation during a-adrenoceptor blockade (P + B). The dilator response to PAN stimulation was enhanced when the constrictor response was attenuated by phentolamine (P+B). n = 7-12; *P < 5, **P < 1, ***P < 1.

8 84 A. KHOSHBA TEN AND W. R. FERRELL phentolamine (1-5 M), an al,c2-blocker, prazosin (1-5 M), an al-blocker, or guanethidine, attenuated the constrictor response to nerve stimulation but rauwolscine, an oc2-blocker (1- M), and a,,f-matp (1-5 M), a P2-purinoceptor desensitizer, did not. It is noticeable that injection of phentolamine not only blocked the constrictor response to PAN stimulation almost completely but in some cases a dilator response then emerged (Fig. 3A). The effectiveness of phentolamine and prazosin but not rauwolscine or a,,-matp in reducing the constrictor effect of PAN stimulation on articular blood vessels suggests that the neurotransmitter involved in constriction of these vessels is noradrenaline acting upon a1-adrenoceptors. As shown in Fig. 3B, complete abolition of the constrictor response to nerve stimulation was not achieved as the injections were administered as a bolus and thus did not remain localized. The maximum concentrations that could be administered were limited by the systemic effects generated by these larger doses. However, in a few experiments it was possible to show that intra-articular administration of phentolamine (1-5 M) abolished the constrictor response. Dorsal root stimulation To assess whether the dilator response to PAN stimulation in these experiments was due to activation of afferent C fibres, the dorsal roots of L7 and Si, which include most of afferent fibres arising from sensory receptors in the dorsal aspect of the knee joint capsule (Skoglund, 1956), were stimulated. As illustrated in Fig. 5B, electrical stimulation of L7 induced a monophasic dilator response in articular blood vessels. L7 stimulation was substantially more effective in producing vasodilatation than similar stimulation of the SI root (Fig. 5C) which may indicate that fewer unmyelinated sensory afferent fibres innervating the posterior region of the knee joint travel in the latter. The dilator response due to L7 stimulation was reduced by intra-articular injection of 1 l tg of substance P antagonist D-Pro4,-D-Trp7'9'1- SP411 prior to electrical stimulation of this root (Fig. 5B and C). The magnitude of the dilator response to L7 stimulation was substantially greater than that occurring during PAN stimulation. This was likely to be due to activation of efferent sympathetic fibres in PAN, as the dilator response to PAN stimulation was somewhat greater when an a-blocker was used (Fig. 5 C). As in many cases it was not possible to completely block the constrictor response to PAN stimulation during a- adrenoceptor blockade (see Fig. 3B), it is likely that the dilator responses observed in response to PAN stimulation during a-adrenoceptor blockade was consequently attenuated and thus were smaller than those occurring during L7 stimulation. DISCUSSION Stimulation of PAN resulted in an initial vasoconstriction of articular blood vessels of all animals (fourteen cats) during the stimulation period followed by a prolonged vasodilatation on cessation of stimulation in eight cats. The results of the present investigation indicate that, consistent with the findings of Cobbold & Lewis in the dog (1956), and Khoshbaten & Ferrell in the rabbit (199), articular nerves such as PAN in the cat also contain sympathetic efferent fibres which are vasoconstrictor in nature. The dilator response was observed in some animals which corresponds with the findings of Ferrell & Cant (1987), but not with those of Cobbold

9 NEURAL REGULATION OF KNEE JOINT BLOOD FLOW & Lewis (1956) where dilator responses to nerve stimulation were not described. In the present experiments the dilator response was shown to arise from afferent fibres as it could be elicited on electrical stimulation of the appropriate dorsal root (L7). The lack of dilator response in the experiments of Cobbold & Lewis (1956) is perhaps due to differences in the techniques, species, or more likely differences in the nerve used (MAN vs. PAN). The present results have confirmed and extended the observations of Ferrell & Cant (1987). Direct comparisons between the two series of experiments is difficult due to differences in the techniques used in these experiments. In the present research the laser Doppler technique sampled very localized areas of the posterior capsule whereas in the experiments of Ferrell & Cant (1987) intraarticular temperature changes were used as an indicator of changes in blood flow. In experiments where the dilator response was absent, the possibility exists that the prolonged recovery period following vasoconstriction induced by PAN stimulation could mask the dilator response. These results also show that like other vascular beds, articular blood vessels are innerved by sympathetic efferent fibres. The vasoconstrictor response to electrical nerve stimulation (PAN) was significantly reduced almost equally effectively by the a1sa2-blocker phentolamine, by guanethidine, and by the al-blocker prazosin, suggesting this response is mainly mediated by noradrenaline. The vasoconstrictor response to PAN stimulation in the cat appeared to be resistant to rauwolscine, an a2-adrenoceptor antagonist. These results are consistent with the previous in vitro observation in rabbits that noradrenaline released from sympathetic efferent fibres is acting mainly via ax-adrenoceptors (Ferrell & Khoshbaten, 199). In the present study no evidence was obtained to indicate that ATP is released from nerve endings and contributes towards the vasoconstriction elicited by electrical stimulation of PAN as pre-treatment with the P2-purinoceptor desensitizer, a, fl-methylene ATP, did not change the magnitude of constrictor response. The findings from this study, taken together with the results of previous experiments (Ferrell & Russell, 1986; Ferrell & Cant, 1987; Khoshbaten & Ferrell, 199), indicate that the components of neurogenic inflammation - vasodilatation and increased permeability of blood vessels - occur on antidromic stimulation of afferent C fibres in articular nerves. It is therefore possible that these fibres could contribute to the initiation or maintenance of joint inflammation. This hypothesis is supported by evidence of a neurogenically mediated component of acute joint inflammation in the rat (Lam & Ferrell, 1989). Intra-articular injection of a substance P antagonist reduced the magnitude of dilator response elicited by either electrical stimulation of dorsal roots (L7) or PAN stimulation. Assuming that D-Pro4-D-Trp7 9' lo-sp4-1is a relatively specific substance P antagonist, it would appear that the dilator response is mediated by articular afferents which release substance P or a related neurokinin from their terminals when depolarized. Therefore, besides their well-known role in nociception, these fibres may have an important role to play in the local regulation of articular blood flow and may perhaps contribute to the pathogenesis of inflammatory joint disease. Financial support from the Wellcome Trust is gratefully acknowledged. 85

10 86 A. KHOSHBA TEN AND W. R. FERRELL REFERENCES BURNSTOCK, G. (1975). Control of smooth muscle activity in vessels by adrenergic nerves and circulating catecholamines. In Smooth Muscle Pharmacology and Physiology, vol. 5, pp INSERM, Paris. BURNSTOCK, G. (198). Cholinergic and purinergic regulation of blood vessels. In Handbook of Physiology, section 2, The Cardiovascular System, vol. ii, Vascular Smooth Muscle, ed. BoHR, D. F., SOMLYO, A. P. & SPARKS, H. V., pp American Physiology Society, Bethesda. BURNSTOCK, G. (1985). Nervous control of smooth muscle by transmitters, cotransmitters and modulators. Experientia 41, BURNSTOCK, G., CHANLEY, J. & CAMPBELL, G. R. (1986). The innervation of arteries. In Structure and Function of the Circulation, vol. 1, ed. SCHWARTZ, C. J., WERTHESSEN, N. T. & WOLF, S., pp Plenum Press, New York. COBBOLD, A. F. & LEWIS,. J. (1956). The nervous control of joint blood vessels. Journal of Physiology 133, COUTURE, R. & CUELLO, A. C. (1984). Studies on the trigeminal antidromic vasodilatation and plasma extravasation in the rat. Journal of Physiology 346, FERRELL, W. R. & CANT, R. (1987). Vasodilatation of articular blood vessels induced by antidromic stimulation of articular C fibre afferents. In Fine Afferent Fibres and Pain, ed. SCHMIDT, R. F., SCHAIBLE, H.-G. & VAHLE-HINz, C. VCH, Weinheim. FERRELL, W. R. & KHOSHBATEN, A. (199). Responses of blood vessels in the rabbit knee to electrical stimulation of the joint capsule. Journal of Physiology 423, FERRELL, W. R. & RUSSELL, N. J. W. (1986). Extravasation in the knee induced by antidromic stimulation of articular C fibre afferents of the anaesthetized cat. Journal of Physiology 379, FREEMAN, M. A. R. & WYKE, B. (1967). The innervation of the knee joint. Anatomical and histological study in cat. Journal of Anatomy 11, GARDNER, E. D. (1944). The distribution and termination of nerves in the knee joint of the cat. Journal of Comparative Neurology 8, KHOSHBATEN, A. & FERRELL, W. R. (199). Responses of blood vessels in the rabbit knee to acute joint inflammation. Annals of the Rheumatic Diseases (in the Press). LAM, F. Y. & FERRELL, W. R. (1989). Inhibition of carrageenan-induced joint inflammation by substance P antagonist. Annals of the Rheumatic Diseases 48, LANGFORD, L. A. & SCHMIDT, R. F. (1983). Afferent and efferent axons in the medial and posterior articular nerves of the cat. Anatomical Record 26, PHILLIPS, D. S. (1978). Basic Statistics for Health Science Students. W. H. Freeman & Company, San Francisco. SATO, Y., SCHAIBLE, H.-G. & SCHMIDT, R. F. (1983). Types of afferents from the knee joint evoking sympathetic reflexes in cat inferior cardiac nerves. Neuroscience Letters 39, SKOGLUND, S. (1956). Anatomical and physiological studies of knee joint innervation in the cat. Acta physiologica scandinavica 36, suppl. 124, 1-11.