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Neurogenic cutaneous vasodilation in the cat forepaw
Journal of the Autonomic Nervous System, 37 (1992) 39-46
39
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01228
Neurogenic cutaneous vasodilation in the cat forepaw Masahiro Kawarai * and Michael C. Koss Department of Pharmacology, Universityof Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, U.S.A. (Received 5 June 1991) (Revision received 6 September 1991) (Accepted 9 September 1991)
Key words: Laser Doppler flowmetry; Preganglionic sympathetic; Cutaneous blood flow; Sudomotor; Atropine; Anti-adrenergic drugs Abstract The present experiments were undertaken to determine, using Laser Doppler flowmetry, if elimination of efferent constrictor mechanisms would unmask cutaneous vasodilator responses following preganglionic sympathetic nerve stimulation in the forepaw of anesthetized cats. We also addressed the question of a potential causal relationship between neurally evoked vasodilator and sudomotor responses. Three separate anti-adrenergic regimens were utilized: (1) acute guanethidine administration (1-2 mg/kg); (2) chronic monoamine depletion with reserpine (5 mg/kg) and a-methyl-para-tyrosine (2 × 300 mg/kg); and (3) a-adrenoceptor blockade with prazosin (300 ~ g / k g ) and yohimbine (0.5 mg/kg). Guanethidine treatment produced a significant depression of basal cutaneous blood flow whereas a-adrenoceptor blockade did not. In all three groups, stimulation of the preganglionic thoracic sympathetic nerve trunk produced intensity-dependent increases of digital skin blood flow along with near-maximal sympatheticcholinergic sudomotor (electrodermal) responses recorded simultaneously from the same paw. Vasodilator responses were not altered by intravenous propranolol (1 mg/kg) or atropine (1 mg/kg); however, evoked sudomotor responses were totally blocked by atropine. Low doses (1.5 mg/kg i.v.) of hexamethonium selectively abolished the cutaneous vasodilator responses but not concomitantly evoked sudomotor responses. These results demonstrate, using direct measurements of blood flow, that cutaneous digital vasodilation can be measured in cats following removal of vasoconstrictor mechanisms either pre- or postjunctionally. Neither muscarinic nor /3-adrenoceptor mechanisms appear to be involved. These experiments also suggest that cutaneous vasodilation is not a consequence of concomitant sudomotor activation.
Introduction
It is widely accepted that sympathetic nerve stimulation generally produces constriction of cu-
Correspondence: M.C. Koss, Department of Pharmacology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190, U.S.A. * Present address: Department of Biosciences, The Nishi Tokyo University, 2525 Uenohara, Yamanashi 409-01, Japan.
taneous blood vessels. However, in dogs and cats that have been pretreated with noradrenergic neuron blocking agents, stimulation of the sympathetic nervous system produces vasodilation in the hind paw pads [1,2,4,6,20,22]. Although there is considerable variability in species and techniques utilized (see Discussion), the overall consensus of these studies is that neurogenic sympathetic vasodilation is observed in both muscle and skin when adrenergic vasoconstrictor mechanisms are eliminated. The increased blood flow in skeletal muscle appears to be primarily choliner-
4{)
gic (muscarinic), whereas residual cutaneous vasodilation is not. Other investigators have speculated that cutaneous vasodilation might be a result of local hyperemia associated with sudomotor activation and that there is not a separate cutaneous vascular vasodilator innervation [7,16]. We have utilized Laser Doppler flowmetry techniques to assess the relative contribution of postjunctional adrenoceptors in neurally evoked cutaneous vasoconstrictor responses in the forelimb of anesthetized cats [15]. Our results clearly demonstrated that both al- and az-adrenoceptots are present in the cutaneous bed of the cat and that the predominant vasoconstrictor effect is mediated by innervated a2-adrenoceptors (i.e., primarily blocked by a2-adrenoceptor antagonists such as yohimbine and rauwolscine) [15]. We have also demonstrated the usefulness of Laser Doppler flowmetry in investigating vascular responses in a cutaneous bed of the cat [14,15]. The present study was undertaken in order to determine if elimination of adrenergic mechanism by either pre- or postjunctional blockade would produce residual vasodilator responses to preganglionic sympathetic stimulation as assessed by Laser Doppler flowmetry. Using this technique we also directly addressed the possible relationship between sudomotor and cutaneous vasodilator mechanisms.
Materials and Methods
Adult cats of either sex (1.8-4.0 kg) were anesthetized with a-chloralose (60-80 m g / k g i.p.). The trachea was intubated for ventilation with a Harvard respirator using room air. A femoral artery and vein were cannulated for recording of systemic arterial blood pressure (Statham P23 pressure transducer) and for the intravenous administration of drugs. Heart rate was derived from the femoral arterial pulse and processed using a cardiotachograph (Grass 7P4). A Grass model 7 polygraph was used to record all physiological parameters. Body temperature was maintained at approximately 37°C by use of a heat lamp placed above the animal. In order to minimize alterations in the paw temperature, the dor-
sal and lateral surfaces were completely wrapped with a thermostable chemical heating pad (Deltaphase Isothermal Pad; Braintree Scientific Inc., Braintree, MA). Cutaneous blood flow measurements were made from a metacarpal toe pad (forepaw). Sudomotor (electrodermal) responses were recorded from the central footpad of the same paw using Beckman miniature biopotential electrodes (11 mm diameter). Details concerning recording and activation of these sympatheticcholinergic skin potential responses in cats have been published [13]. Sympathetic nerL,e stimulation
For preganglionic sympathetic nerve stimulation, ribs 2-4 were tied and sectioned for exposure of the thoracic sympathetic chain. The sympathetic nerve trunk was crushed proximally and a pair of silver wire stimulating electrodes was placed under the nerve trunk caudal to the stellate ganglion with the stimulation site immersed in warm mineral oil. Square wave pulses of supramaximal voltage and duration (6-10 V and 1-2 ms) were derived from a Grass $88 stimulator and Grass SIU5 isolation unit. Usually, 7-10 s trains of stimuli were applied with the frequency varied from 0.25 to 16 Hz. The cats were treated with blocking agents in order to reduce adrenergically mediated vasoconstriction as follows: (1) cats pretreated acutely with guanethidine sulfate (1-2 m g / k g i.v.); (2) animals treated with combined prazosin (300 /~g/kg i.v.) and yohimbine (0.5 m g / k g i.v.); and (3) preparations depleted of monoamines with reserpine (5 m g / k g i.p.) 24 h before, and a synthesis inhibitor, c~-methyl-para-tyrosine (300 m g / k g i.p.), given with the reserpine and again 2 h before the start of experimentation. Due to a potent 'first dose' effect, prazosin was administered gradually in increasing cumulative doses from 3-300 p,g/kg. In monoamine depleted animals the dose of anesthetic was reduced by 50%. In some of the depleted experiments, the extent of removal of noradrenergic mechanisms was determined using intraarterial (i.a.) injections of tyramine. For i.a. injection, blood was diverted from the right carotid artery into the right brachial artery (at the midpoint of the forelimb). Details
41 concerning i.a. injection to the digital cutaneous vascular bed of the cat have been published [14]. After establishing the frequency-response relationship, a frequency of stimulation that produced a 50-75% of the peak activation in cutaneous blood flow was utilized (generally 1-5 Hz). Under these conditions electrodermal response (EDR) amplitudes were usually maximal [13]. Antagonists were administered intravenously with 10-15 min allowed to reach steady state.
group. Changes of EDR amplitude and cutaneous blood flow, before and after antagonist administration, were analyzed using Student's ttest for paired comparisons with significance accepted at the P < 0.05 level. Generally, three control and three responses taken after antagonist administration were averaged.
Assessment of cutaneous blood flow changes In most experiments cutaneous blood flow was determined by Laser Doppler flowmetry using a Perimed Pf2 Laser Doppler flowmeter fitted with a PF103 fiber optic probe (one emission and 2 receiving fibers in 2.5-mm diameter tip). In the other experiments a Perimed Pf3 Laser Doppler flowmeter fitted with a PF310 fiber optic probe (1-mm diameter) was used. The results obtained were similar and are reported together. The principles and application of Laser Doppler flowmetry recently have been summarized [14,15,21]. As this technique gives only relative flow values, the data are reported as the area under the vasodilatot curves in arbitrary units. The level of zero blood flow was determined in each experiment after sacrifice with pentobarbital. One major disadvantage of this methodology is that only animals with non-pigmented ('pink') footpads can be utilized.
Guanethidine treatment Cutaneous vasoconstrictor and electrodermal responses were elicited in nine cats before, during and after administration of guanethidine (1-2 mg/kg i.v.). In five animals a single 1 mg/kg dose was sufficient to almost totally abolish the neurally evoked cutaneous vasoconstrictor response. Two animals required a second guanethidine injection (two maintained a slight vasoconstrictor response even after the second dose of guanethidine). In four experiments an obvious vasodilator response was spontaneously unmasked following acute guanethidine treatment (Fig. 1); in two, neurally evoked elevation of blood flow was observed only when the intensity of stimulation was increased, and in three cats, no appreciable vasodilation was elicited. Fig. 1 is an example of preganglionically evoked responses and the subsequent effects of i.v. guanethidine. Sympathetic-cholinergic sudomotor (electrodermal) responses were near maximal and were significantly depressed by guanethidine treatment (6.4 + 0.8 before and 4.8 + 0.6 mV after; P < 0.01). Mean blood pressure also was depressed by drug treatment from 112 + 9 to 80 + 12 mmHg (P < 0.01), although heart rate was not significantly altered (220 + 11 to 201 + 13 beats/min). More importantly, basal cutaneous blood flow was depressed by this treatment regimen (from 916 + 140 to 651 + 139 mV; P < 0.5). Depression of basal cutaneous flow ranged from 7 to 69% of control.
Drugs and statistics All drug solutions were prepared in physiological saline with the exception of yohimbine (distilled water) and prazosin [2.5% glucose (w/v): 2.5% glycerol (v/v)]. Drug dosages refer to the respective salts. The following drugs were used: (+)-propranolol hydrochloride, yohimbine hydrochloride, hexamethonium bromide (C6) , and amethyl-para-tyrosine methylester hydrochloride (Sigma Chemical Co., St. Louis, MO); atropine sulfate (Nutritional Biochemicals, Cleveland, OH); guanethidine sulfate and reserpine (Serpasil) (Ciba Pharmaceutical Co., Summit, N J); prazosin hydrochloride (Pfizer Inc., Groton, CT). Data are reported as means + SEM with n representing the number of experiments in each
Results
Monoamine depletion Seven cats were depleted of monoamines with reserpine and a synthesis inhibitor. Basal blood pressure was 90 + 9 mmHg and heart rate was
42 1 8 t _ 9 b e a t s / m i n . Preganglionic sympathetic nerve stimulation readily evoked responses from the sympathetic-cholinergic s u d o m o t o r system which were maximal in amplitude at all stimulation frequencies used. In four cats, nerve stimulation resulted in vasodilator responses which were frequency d e p e n d e n t with peak activation occurring between 2 and 4 Hz. In one preparation, a modest vasoconstrictor response p r e c e d e d an elevation of cutaneous blood flow, and in two no response over the spontaneous fluctuations in skin blood flow was obtained. A l t h o u g h p r e t r e a t m e n t m e a s u r e m e n t s were not possible, basal cutaneous blood flow levels a p p e a r e d to be lower than is our experience with n o n - p r e t r e a t e d controls (similar to values as seen after guanethidine treatment). Intraarterial tyramine (10/zg) p r o d u c e d a modest vasoconstrictor response in the three preparations tested (18 _+
13% of maximal). In five non-treated controls, tyramine (10 /xg i.a.) decreased cutaneous blood flow by 64 _+ 9%.
a-Adrenoceptor antagonism The third treatment regimen was to stimulate the preganglionic sympathetic nerve trunk to obtain vasoconstrictor responses of 4 0 - 6 0 % of maximal [15], Seven animals were then challenged (Fig. 2) with increasing doses of prazosin ( 3 - 3 0 0 / ~ g / k g i.v.) followed by a single injection of yohimbine (0.5 m g / k g i.v.). Blood pressure was reduced by about 20 m m H g but neither heart rate nor basal cutaneous blood flow was significantly altered. Evoked s u d o m o t o r response amplitude also was not affected by cq- and a 2a d r e n o c e p t o r blockade. In three of these seven preparations, c~-adrenoceptor blockade resulted in a reversal of the vasoconstrictor to a vasodila-
A
1 min
CBF 1H z
1 Hz
2eHZ
,Hz
Guanethidine l m g / k g
0
B
C
EDR
CBF 1 •Hz
2 •Hz
4 •Hz
,~
l~z
Guanethidine 1 mg/kg
Fig. 1. Electrodermal (EDR) and cutaneous blood flow (CBF) responses in a chloralose anesthetized cat evoked by electrical stimulation (dots) of the thoracic preganglionic nerve trunk. In (A) the frequency of stimulation was held at I Hz (10 V; 7-s train; 2 ms pulses). Guanethidine (1 mg/kg i.v.) was infused as shown (solid bar); (B) Frequency varied between 1-4 Hz 10 min after guanethidine; (C) Frequency-response 10 min after second dose of guanethidine (1 mg/kg i.v.).
43 looo CBF
A
1ram
1m~
EDR
Prazo6in(101Jglkg) Ii
CBF
Prazo6in[30 ug/kg)
Hexamethonlum(1.5mg/kg)
Prazosin(100IJg/kg) B
l min
Prazo~in1300Iaglkg)
EDR
Yohimtwne10.5mg/kg) 1i
Fig. 2. Vasoconstrictor cutaneous blood flow (CBF) responses to preganglionic sympathetic nerve stimulation in an anesthetized cat (6 V; 4 Hz; 10-s train; 2 ms pulses). Stimuli presented at approximately 3-min intervals. Intravenous prazosin was administered in cumulative doses (10-300 /zg/kg) at 10-15 min intervals followed by a single injection of yohimbine (0.5 mg/kg i.v.). Note that prazosin produced only modest inhibition of nerve-evoked vasoconstrictor response with total abolition of constrictor responses after yohimbine.
Hexamethonium(1.5mg/kg)
Fig. 4. Polygraph recording of electroderma] (EDR) and cutaneous blood flow (CBF) responses to preganglionic sympa-
thetic nerve stimulation in two anesthetized cats previously pretreated with prazosin and yohimbine (as in Fig. 3). Hexamethonium (1.5 mg/kg i.v.) was administered at arrows. (A) Pure vasodilator response (6 V; 4 Hz; 7-s train; 1 ms pulse). (B) Mixed vasoconstrictor-vasodilator response (9 V; 4 Hz; 7-s train; 1 ms pulses). Note blockade of increased blood flow in both cases with no significant effect on evoked sudomotor responses and no blockade of vasoconstrictor component of response.
tor response that ranged from 6-60% of the control amplitude. In two cats, increased skin blood flow was observed only when the intensity o f s t i m u l a t i o n w a s i n c r e a s e d . Figs. 2 a n d 3 illustrate a typical response pattern. Of the remaining two animals, one exhibited only vasoconstriction f o l l o w i n g n e r v e s t i m u l a t i o n a n d i n o n e , n o significant vasomotor response was elicited after aadrenoceptor blockade.
I~
CBF
Response stability and autonomic blockade Once established, cutaneous vasodilator responses were quite stable when evoked repeate d l y o v e r t i m e (Fig. 4). E v o k e d s u d o m o t o r a n d
CBF
0 0.5 HZ
16 Hz
__~ 1 HZ
16 Hz
2 Hz
4 Hz
16 Hz ~, 6 Hexamethonium (20 mg/kg)
8 Hz
16 Hz
Fig. 3. Continuation of cutaneous blood flow (CBF) responses from Fig. 2 (after prazosin and yohimbine). The intensity of stimulation was increased from 6 to 9 V, which in turn, unmasked a residual vasodilator response. Note that, as the frequency of stimulation increased, mixed vasoconstrictor and vasodilator responses are manifest. Administration of the ganglionic blocking drug, hexamethonium (20 mg/kg i.v.), totally abolished all responses to preganglionic nerve stimulation.
44
"J~
EDR
mal responses. A higher dose of C 6 (20 m g / k g i.v.; data not shown) totally eliminated all nerve evoked responses.
(mY) i
Discussion
2
0
251
I l
(U ~) 20 15 i
0
Prop Atropine
C~
Fig. 5. Composite representation of electrodermal (EDR) and cutaneous vasodilator (CBF) responses due to preganglionic sympathetic nerve stimulation before and after treatment with autonomic blocking agents in anesthetized cats. Solid bars, control responses to sympathetic nerve stimulation. Crosshatched bars, magnitude of responses 10 to 15 rain after i.v. administration of propranolol (prop; 1 m g / k g ) , atropine (1 m g / k g ) or hexametbonium (C6; 1.5 m g / k g ) . The vasodilator responses are expressed in arbitrary units (U 2) which represent area under the vasodilator curve. For the propranolol group, three cats were depleted of monoamines with reserpine and a-n,ethyl-p-tyrosine and one was pretreated with yohimbine and prazosin. In the experiments using atropine, one cat was depleted of monoamines and two were pretreated with yohimbine and prazosin. For hexamethonium, two cats were m o n o a m i n e depleted, two were pretreated with guanethidine and two were a-adrenoceptor blocked. Values are m e a n s + S E M . * * P < 0.01. Stimulation parameters: 6 to 10 V: I - 2 ms pulse duration: 7-10-s trains presented at 2- to 3-min intervals.
vasodilator responses were observed before and after blockade with i.v. propranolol (1 mg/kg), atropine (1 m g / k g ) and two different dosages (1.5 and 20 mg/kg) of hexamethonium (C6). As shown (Figs. 4 and 5) neither /3-adrenergic nor muscarinic receptor blockade altered neurally evoked cutaneous vasodilation. The sudomotor (EDR) system was almost totally blocked by atropine but not affected by propranolol. With regard to ganglionic blockade, the low dose of C 6 (1.5 m g / k g i.v.) almost completely eliminated the vasodilator responses while having no significant action on the simultaneously evoked electroder-
In the present study, preganglionic sympathetic nerve evoked responses (forepaw cutaneous blood flow alterations as measured by Laser Doppler flowmetry and neurally elicited sudomotot responses) were studied in chloralose anesthetized cats. In non-pretreated animals, nerve stimulation produced near maximal sympatheticcholinergic electrodermal responses and frequency-related cutaneous vasoconstrictions. The observation that vasoconstrictor responses were selectively abolished by combined a~- and c~2adrenoceptor antagonism (with the predominant effect being a2-adrenoceptor mediated) is in total agreement with our previous reports [14,15]. Others also have recently demonstrated a predominance of innervated posqunctional a2-adrenoceptors in small terminal arterioles in vivo [19]. Most studies in this area have used dogs pretreated with guanethidine [1,2,6,20,22], although cats also have been studied [4,22]. In addition to guanethidine, reserpine and bretylium have been utilized for anti-adrenergic effects [1,20,22]. Cutaneous flow was measured indirectly by subtraction from total limb blood flow by skinning or occlusion of the paw [2,20,22], by direct measurement of perfusion pressure in small arteries [1,6], or by measurement of surface temperature changes in the footpad of the cats [4]. The overall consensus of these studies is that sympathetic nerve stimulation produces an active vasodilator response in cutaneous blood vessels of the paw by action on receptors that are non-cholinergic, non-adrenergic and non-histaminergic. There also is evidence that the cutaneous vasodilator response is not mediated by bradykinin [1,2], /3-adrenergic [1-3,18], or prostaglandin [2] mechanisms. Bell [2,3] believes that dopamine may be the vasodilator mediator; however, the lack of specificity of the antagonists used, along with the presence of active vasodilation following reserpine [1,18] or chronic guanethidine pretreat-
45 merit [4], makes this conclusion less than totally convincing. Recently, calcitonin gene-related peptide has been shown to produce a very large increase in skin blood flow in humans [10] and is, along with a variety of other peptides, a possible candidate for the vasodilatory substance. This question will likely not be resolved until sufficiently specific antagonists for the various putative vasodilators are developed. The present study in cats used a more direct approach (Laser Doppler flowmetry) and compared three different modes of eliminating the noradrenergic vasoconstrictor component. Cutaneous vasodilator responses were unmasked following either prejunctional (monoamine depletion or guanethidine) or postjunctional (aadrenoceptor) antagonism, although not all animals tested exhibited a pronounced vasodilator effect. Difficulty in obtaining consistent vasodilation in adrenergic blocked cats is consistent with the early report by Zimmerman [22] in which he observed non-cholinergic cutaneous vasodilation in less than half of the animals tested. The reason for this variability is not clear although in the present study, there was no apparent relationship with regard to the particular anti-adrenergic regimen used. Of the three dosage regimens used, guanethidine most clearly decreased basal cutaneous blood flow. A similar depressant increase in resistance to flow has been reported with this agent in birds [3,18]. It may be that continuous release of catecholamines is responsible for this effect [9] although a non-specific effect might be present as guanethidine also decreased electrodermal response amplitude. The contribution of this depressant action in generating subsequent vasodilation is unknown. It is of interest, however, that vasodilator responses are manifest after a-adrenoceptor blockade (which by itself did not depress basal skin blood flow). Residual cutaneous vasodilation due to sympathetic nerve stimulation appears to be a general phenomenon as active cutaneous vasodilation is observed after adrenergic neuron blockade in a variety of other species including rats [8], birds [3,18] and humans [11]. In some studies postganglionic nerves or dorsal roots were stimulated
with the potential of retrograde activation of sensory nerve fibers and subsequent release of vasoactive peptides such as substance P [4,8,12]. In the present investigation, as in many others [4,6,17,20,22], the decentralized preganglionic nerve trunk was stimulated. This, along with the sensitivity to hexamethonium, strongly supports activation of sympathetic vasodilator efferent nerve fibers. A high sensitivity to hexamethonium blockade has also been demonstrated for muscle vasoconstrictor responses [5], whereas neurally evoked cutaneous vasoconstrictor responses are resistant to blockade by low doses of hexamethonium [5,15]. It has been proposed that cutaneous vasodilation is not a distinct event, but is produced by vasodilator substances secondary to sudomotor glandular activation [7,16]. The observation that atropine abolishes the sudomotor responses but not the cutaneous vasodilation combined with the clear separation of these neurally evoked events by low doses of hexamethonium, argues against their hypothesis. In addition, active sympathetic evoked cutaneous vasodilation is seen in birds even though they arc devoid of a sudomotor system [3,18]. The present results are totally in agreement with those of Bell et al. [4] in which they performed similar experiments using surface temperature changes as an index of cutaneous blood flow. In their study using guanethidine pre-treated cats they found that atropine selectively blocked sudomotor activation, whereas low doses of hexamethonium selectively inhibited preganglionic evoked temperature elevations. In extending the work by Bell et al. [4], we have demonstrated that active non-cholinergic, non-adrenergic cutaneous vasodilation can be measured in the forepaws of anesthetized cats using Laser Doppler flowmetry. Both pre(guanethidine pretreatment and monoamine depletion) and postjunctional ( a - a d r e n o c e p t o r blockade) elimination of the vasoconstrictor component was effective in unmasking vasodilation although basal cutaneous blood flow is better preserved by a-adrenoceptor blockade. Finally, the selective blockade by atropine and low doses of hexamethonium argue against a causal linkage
46
between cutaneous vasodilation and sudomotor
activation.
Acknowledgements The authors thank Mrs. Linda Hess for her expert technical assistance and Ms. H. Whiteside for preparing the manuscript. We are grateful to KAO Corporation for financial assistance.
References 1 Ballard, D.R., Abboud, F.M, and Mayer, H.E., Release of a humoral vasodilator substance during neurogenic vasodilatation, Am. J. Physiol., 219 11970) 1451-1457. 2 Bell, C. and Lang, W.J., Evidence for dopaminergic w~sodilator innervation of the canine paw pad, Br. J. Pharmacol., 67 11979) 337-343. 3 Bell, C. and Rome, A.C., Pharmacological investigations of the vasodilator nerves supplying the duck's foot, Br. J. Pharmacol., 82 11984) 801-808. 4 Bell, C., J~inig, W., Kiimmel, H. and Xu, H., Differentiation of vasodilator and sudomotor responses in the cat paw pad to preganglionic sympathetic stimulation, J. Physiol., 364 (1985) 93-104. 5 Bell, C., Differentiation of neurogenic vasoconstrictor responses in skin and skeletal muscle of dog hindlimb, J. Auton. Nerv. Syst., 14 11985) 13-21. ~ Brody, M.J. and Shaffer, R.A., Distribution of vasodilator nerves in the canine hindlimb, Am. J. Physiol., 218 (19711) 470-474. 7 Fox, R.tt. and Hilton, S.M., Bradykinin formation in human skin as a factor in heat vasodilatation, J. Physiol., 142 (1958) 219-232. 8 Gamse, R. and Saria, A., Antidromic vasodilatation in the rat hindpaw measured by laser Doppler flowmetry: pharmacological modulation, J. Auton. Nerv. Syst., 19 (1987) 11)5-11 t. 9 Gillespie, J.S., The rat anococcygeus muscle and its response to nerve stimulation and to some drugs, Br. J. Pharmacol., 45 (1972) 4/)4-416.
10 Jiiger, K., Muench, R., Seifert, H., Beglinger, C., Bollinger, A. and Fischer, J.A., Calcitonin gene-related peptide (CGRP) causes redistribution of blood flow in humans, Eur. J. Clin. Pharmacol., 39 (19911) 401-494. I 1 Kellogg, D.L., Jr., Johnson, J,M. and Kosiba, W.A., Selective abolition of adrenergic vasoconstrictor responses in skin by local iontophoresis of bretylium, Am. J. Physiol., 257 (1989) H1599-HI61)6. 12 Khoshbaten, A. and Ferrell, W R . , Alterations in cat knee joint blood flow induced by electrical stimulation of articular afferents and efferents, J. Physiol., 430 (199/)) 77-86. 13 Koss. M.C. and Davison, M.A., Characteristics of lhe electrodermal response: A model for analysis of central sympathetic reactivity, Naunyn-Schmiedeberg's Arch. Pharmacol., 295 (1976) 153-158. 14 Koss, M.C., Characterization of adrenoceptor subtypes in cat cutaneous vasculature, J. Pharmacol. Exp. Ther.. 254 (1990) 221-227. 15 Koss, M.C., Kawarai, M. and lto, T., Neural activation of alpha-2 adrenoceptors in cat cutaneous vasculature, J. Pharmacol. Exp. Ther., 256 (1991) 1126 1131. 1~'~ Love, A.H.G. and Shanks, R.G., The relationship between the onset of sweating and vasodilatation in the forearm during body heating, J. Physiol., 162 11962) 121- 128. 17 l,undberg, J., Norgren, L., Ribbe, E., Ros~n, I., Steen, S., Th6rne J. and Wallin, 13.G., Direct evidence of active sympathetic vasodilatation in the skin of the h u m a n fool, J. Physiol.. 417 (1989) 437-446. 18 McGregor, D.D., Noncholinergic vasodilator innervation "3 in the feet of the ducks and chickens, Am. ,1. Physiol..,37 11979) Hl12-11117. I t) Ohyanagi, M., Faber, J.E. and Nishigaki, K., Differential activation of cry- and ~e2-adrenoceptors on microvascular smooth muscle during sympathetic nerve stimulation, Circ. Res., 68 (1991) 232-244. 2(1 Rolewicz, T.F. and Zimmerman, B.('., Peripheral distribution of cutaneous sympathetic vasodilator system, Am. J. Physiol., 223 (1972) 939-943. 21 Shepherd, A.P. and Oberg, P.A., Laser-Doppler blood l]owmetry, Kluwer Academic Publishers, Boston, 1990. 22 Zimmerman, B.G.. Comparison of sympathetic vasodilator innervation of hindlimb of the dog and cat, Am. J. Physiol.. 214 11968) 62-66.
39
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00 JANS 01228
Neurogenic cutaneous vasodilation in the cat forepaw Masahiro Kawarai * and Michael C. Koss Department of Pharmacology, Universityof Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, U.S.A. (Received 5 June 1991) (Revision received 6 September 1991) (Accepted 9 September 1991)
Key words: Laser Doppler flowmetry; Preganglionic sympathetic; Cutaneous blood flow; Sudomotor; Atropine; Anti-adrenergic drugs Abstract The present experiments were undertaken to determine, using Laser Doppler flowmetry, if elimination of efferent constrictor mechanisms would unmask cutaneous vasodilator responses following preganglionic sympathetic nerve stimulation in the forepaw of anesthetized cats. We also addressed the question of a potential causal relationship between neurally evoked vasodilator and sudomotor responses. Three separate anti-adrenergic regimens were utilized: (1) acute guanethidine administration (1-2 mg/kg); (2) chronic monoamine depletion with reserpine (5 mg/kg) and a-methyl-para-tyrosine (2 × 300 mg/kg); and (3) a-adrenoceptor blockade with prazosin (300 ~ g / k g ) and yohimbine (0.5 mg/kg). Guanethidine treatment produced a significant depression of basal cutaneous blood flow whereas a-adrenoceptor blockade did not. In all three groups, stimulation of the preganglionic thoracic sympathetic nerve trunk produced intensity-dependent increases of digital skin blood flow along with near-maximal sympatheticcholinergic sudomotor (electrodermal) responses recorded simultaneously from the same paw. Vasodilator responses were not altered by intravenous propranolol (1 mg/kg) or atropine (1 mg/kg); however, evoked sudomotor responses were totally blocked by atropine. Low doses (1.5 mg/kg i.v.) of hexamethonium selectively abolished the cutaneous vasodilator responses but not concomitantly evoked sudomotor responses. These results demonstrate, using direct measurements of blood flow, that cutaneous digital vasodilation can be measured in cats following removal of vasoconstrictor mechanisms either pre- or postjunctionally. Neither muscarinic nor /3-adrenoceptor mechanisms appear to be involved. These experiments also suggest that cutaneous vasodilation is not a consequence of concomitant sudomotor activation.
Introduction
It is widely accepted that sympathetic nerve stimulation generally produces constriction of cu-
Correspondence: M.C. Koss, Department of Pharmacology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190, U.S.A. * Present address: Department of Biosciences, The Nishi Tokyo University, 2525 Uenohara, Yamanashi 409-01, Japan.
taneous blood vessels. However, in dogs and cats that have been pretreated with noradrenergic neuron blocking agents, stimulation of the sympathetic nervous system produces vasodilation in the hind paw pads [1,2,4,6,20,22]. Although there is considerable variability in species and techniques utilized (see Discussion), the overall consensus of these studies is that neurogenic sympathetic vasodilation is observed in both muscle and skin when adrenergic vasoconstrictor mechanisms are eliminated. The increased blood flow in skeletal muscle appears to be primarily choliner-
4{)
gic (muscarinic), whereas residual cutaneous vasodilation is not. Other investigators have speculated that cutaneous vasodilation might be a result of local hyperemia associated with sudomotor activation and that there is not a separate cutaneous vascular vasodilator innervation [7,16]. We have utilized Laser Doppler flowmetry techniques to assess the relative contribution of postjunctional adrenoceptors in neurally evoked cutaneous vasoconstrictor responses in the forelimb of anesthetized cats [15]. Our results clearly demonstrated that both al- and az-adrenoceptots are present in the cutaneous bed of the cat and that the predominant vasoconstrictor effect is mediated by innervated a2-adrenoceptors (i.e., primarily blocked by a2-adrenoceptor antagonists such as yohimbine and rauwolscine) [15]. We have also demonstrated the usefulness of Laser Doppler flowmetry in investigating vascular responses in a cutaneous bed of the cat [14,15]. The present study was undertaken in order to determine if elimination of adrenergic mechanism by either pre- or postjunctional blockade would produce residual vasodilator responses to preganglionic sympathetic stimulation as assessed by Laser Doppler flowmetry. Using this technique we also directly addressed the possible relationship between sudomotor and cutaneous vasodilator mechanisms.
Materials and Methods
Adult cats of either sex (1.8-4.0 kg) were anesthetized with a-chloralose (60-80 m g / k g i.p.). The trachea was intubated for ventilation with a Harvard respirator using room air. A femoral artery and vein were cannulated for recording of systemic arterial blood pressure (Statham P23 pressure transducer) and for the intravenous administration of drugs. Heart rate was derived from the femoral arterial pulse and processed using a cardiotachograph (Grass 7P4). A Grass model 7 polygraph was used to record all physiological parameters. Body temperature was maintained at approximately 37°C by use of a heat lamp placed above the animal. In order to minimize alterations in the paw temperature, the dor-
sal and lateral surfaces were completely wrapped with a thermostable chemical heating pad (Deltaphase Isothermal Pad; Braintree Scientific Inc., Braintree, MA). Cutaneous blood flow measurements were made from a metacarpal toe pad (forepaw). Sudomotor (electrodermal) responses were recorded from the central footpad of the same paw using Beckman miniature biopotential electrodes (11 mm diameter). Details concerning recording and activation of these sympatheticcholinergic skin potential responses in cats have been published [13]. Sympathetic nerL,e stimulation
For preganglionic sympathetic nerve stimulation, ribs 2-4 were tied and sectioned for exposure of the thoracic sympathetic chain. The sympathetic nerve trunk was crushed proximally and a pair of silver wire stimulating electrodes was placed under the nerve trunk caudal to the stellate ganglion with the stimulation site immersed in warm mineral oil. Square wave pulses of supramaximal voltage and duration (6-10 V and 1-2 ms) were derived from a Grass $88 stimulator and Grass SIU5 isolation unit. Usually, 7-10 s trains of stimuli were applied with the frequency varied from 0.25 to 16 Hz. The cats were treated with blocking agents in order to reduce adrenergically mediated vasoconstriction as follows: (1) cats pretreated acutely with guanethidine sulfate (1-2 m g / k g i.v.); (2) animals treated with combined prazosin (300 /~g/kg i.v.) and yohimbine (0.5 m g / k g i.v.); and (3) preparations depleted of monoamines with reserpine (5 m g / k g i.p.) 24 h before, and a synthesis inhibitor, c~-methyl-para-tyrosine (300 m g / k g i.p.), given with the reserpine and again 2 h before the start of experimentation. Due to a potent 'first dose' effect, prazosin was administered gradually in increasing cumulative doses from 3-300 p,g/kg. In monoamine depleted animals the dose of anesthetic was reduced by 50%. In some of the depleted experiments, the extent of removal of noradrenergic mechanisms was determined using intraarterial (i.a.) injections of tyramine. For i.a. injection, blood was diverted from the right carotid artery into the right brachial artery (at the midpoint of the forelimb). Details
41 concerning i.a. injection to the digital cutaneous vascular bed of the cat have been published [14]. After establishing the frequency-response relationship, a frequency of stimulation that produced a 50-75% of the peak activation in cutaneous blood flow was utilized (generally 1-5 Hz). Under these conditions electrodermal response (EDR) amplitudes were usually maximal [13]. Antagonists were administered intravenously with 10-15 min allowed to reach steady state.
group. Changes of EDR amplitude and cutaneous blood flow, before and after antagonist administration, were analyzed using Student's ttest for paired comparisons with significance accepted at the P < 0.05 level. Generally, three control and three responses taken after antagonist administration were averaged.
Assessment of cutaneous blood flow changes In most experiments cutaneous blood flow was determined by Laser Doppler flowmetry using a Perimed Pf2 Laser Doppler flowmeter fitted with a PF103 fiber optic probe (one emission and 2 receiving fibers in 2.5-mm diameter tip). In the other experiments a Perimed Pf3 Laser Doppler flowmeter fitted with a PF310 fiber optic probe (1-mm diameter) was used. The results obtained were similar and are reported together. The principles and application of Laser Doppler flowmetry recently have been summarized [14,15,21]. As this technique gives only relative flow values, the data are reported as the area under the vasodilatot curves in arbitrary units. The level of zero blood flow was determined in each experiment after sacrifice with pentobarbital. One major disadvantage of this methodology is that only animals with non-pigmented ('pink') footpads can be utilized.
Guanethidine treatment Cutaneous vasoconstrictor and electrodermal responses were elicited in nine cats before, during and after administration of guanethidine (1-2 mg/kg i.v.). In five animals a single 1 mg/kg dose was sufficient to almost totally abolish the neurally evoked cutaneous vasoconstrictor response. Two animals required a second guanethidine injection (two maintained a slight vasoconstrictor response even after the second dose of guanethidine). In four experiments an obvious vasodilator response was spontaneously unmasked following acute guanethidine treatment (Fig. 1); in two, neurally evoked elevation of blood flow was observed only when the intensity of stimulation was increased, and in three cats, no appreciable vasodilation was elicited. Fig. 1 is an example of preganglionically evoked responses and the subsequent effects of i.v. guanethidine. Sympathetic-cholinergic sudomotor (electrodermal) responses were near maximal and were significantly depressed by guanethidine treatment (6.4 + 0.8 before and 4.8 + 0.6 mV after; P < 0.01). Mean blood pressure also was depressed by drug treatment from 112 + 9 to 80 + 12 mmHg (P < 0.01), although heart rate was not significantly altered (220 + 11 to 201 + 13 beats/min). More importantly, basal cutaneous blood flow was depressed by this treatment regimen (from 916 + 140 to 651 + 139 mV; P < 0.5). Depression of basal cutaneous flow ranged from 7 to 69% of control.
Drugs and statistics All drug solutions were prepared in physiological saline with the exception of yohimbine (distilled water) and prazosin [2.5% glucose (w/v): 2.5% glycerol (v/v)]. Drug dosages refer to the respective salts. The following drugs were used: (+)-propranolol hydrochloride, yohimbine hydrochloride, hexamethonium bromide (C6) , and amethyl-para-tyrosine methylester hydrochloride (Sigma Chemical Co., St. Louis, MO); atropine sulfate (Nutritional Biochemicals, Cleveland, OH); guanethidine sulfate and reserpine (Serpasil) (Ciba Pharmaceutical Co., Summit, N J); prazosin hydrochloride (Pfizer Inc., Groton, CT). Data are reported as means + SEM with n representing the number of experiments in each
Results
Monoamine depletion Seven cats were depleted of monoamines with reserpine and a synthesis inhibitor. Basal blood pressure was 90 + 9 mmHg and heart rate was
42 1 8 t _ 9 b e a t s / m i n . Preganglionic sympathetic nerve stimulation readily evoked responses from the sympathetic-cholinergic s u d o m o t o r system which were maximal in amplitude at all stimulation frequencies used. In four cats, nerve stimulation resulted in vasodilator responses which were frequency d e p e n d e n t with peak activation occurring between 2 and 4 Hz. In one preparation, a modest vasoconstrictor response p r e c e d e d an elevation of cutaneous blood flow, and in two no response over the spontaneous fluctuations in skin blood flow was obtained. A l t h o u g h p r e t r e a t m e n t m e a s u r e m e n t s were not possible, basal cutaneous blood flow levels a p p e a r e d to be lower than is our experience with n o n - p r e t r e a t e d controls (similar to values as seen after guanethidine treatment). Intraarterial tyramine (10/zg) p r o d u c e d a modest vasoconstrictor response in the three preparations tested (18 _+
13% of maximal). In five non-treated controls, tyramine (10 /xg i.a.) decreased cutaneous blood flow by 64 _+ 9%.
a-Adrenoceptor antagonism The third treatment regimen was to stimulate the preganglionic sympathetic nerve trunk to obtain vasoconstrictor responses of 4 0 - 6 0 % of maximal [15], Seven animals were then challenged (Fig. 2) with increasing doses of prazosin ( 3 - 3 0 0 / ~ g / k g i.v.) followed by a single injection of yohimbine (0.5 m g / k g i.v.). Blood pressure was reduced by about 20 m m H g but neither heart rate nor basal cutaneous blood flow was significantly altered. Evoked s u d o m o t o r response amplitude also was not affected by cq- and a 2a d r e n o c e p t o r blockade. In three of these seven preparations, c~-adrenoceptor blockade resulted in a reversal of the vasoconstrictor to a vasodila-
A
1 min
CBF 1H z
1 Hz
2eHZ
,Hz
Guanethidine l m g / k g
0
B
C
EDR
CBF 1 •Hz
2 •Hz
4 •Hz
,~
l~z
Guanethidine 1 mg/kg
Fig. 1. Electrodermal (EDR) and cutaneous blood flow (CBF) responses in a chloralose anesthetized cat evoked by electrical stimulation (dots) of the thoracic preganglionic nerve trunk. In (A) the frequency of stimulation was held at I Hz (10 V; 7-s train; 2 ms pulses). Guanethidine (1 mg/kg i.v.) was infused as shown (solid bar); (B) Frequency varied between 1-4 Hz 10 min after guanethidine; (C) Frequency-response 10 min after second dose of guanethidine (1 mg/kg i.v.).
43 looo CBF
A
1ram
1m~
EDR
Prazo6in(101Jglkg) Ii
CBF
Prazo6in[30 ug/kg)
Hexamethonlum(1.5mg/kg)
Prazosin(100IJg/kg) B
l min
Prazo~in1300Iaglkg)
EDR
Yohimtwne10.5mg/kg) 1i
Fig. 2. Vasoconstrictor cutaneous blood flow (CBF) responses to preganglionic sympathetic nerve stimulation in an anesthetized cat (6 V; 4 Hz; 10-s train; 2 ms pulses). Stimuli presented at approximately 3-min intervals. Intravenous prazosin was administered in cumulative doses (10-300 /zg/kg) at 10-15 min intervals followed by a single injection of yohimbine (0.5 mg/kg i.v.). Note that prazosin produced only modest inhibition of nerve-evoked vasoconstrictor response with total abolition of constrictor responses after yohimbine.
Hexamethonium(1.5mg/kg)
Fig. 4. Polygraph recording of electroderma] (EDR) and cutaneous blood flow (CBF) responses to preganglionic sympa-
thetic nerve stimulation in two anesthetized cats previously pretreated with prazosin and yohimbine (as in Fig. 3). Hexamethonium (1.5 mg/kg i.v.) was administered at arrows. (A) Pure vasodilator response (6 V; 4 Hz; 7-s train; 1 ms pulse). (B) Mixed vasoconstrictor-vasodilator response (9 V; 4 Hz; 7-s train; 1 ms pulses). Note blockade of increased blood flow in both cases with no significant effect on evoked sudomotor responses and no blockade of vasoconstrictor component of response.
tor response that ranged from 6-60% of the control amplitude. In two cats, increased skin blood flow was observed only when the intensity o f s t i m u l a t i o n w a s i n c r e a s e d . Figs. 2 a n d 3 illustrate a typical response pattern. Of the remaining two animals, one exhibited only vasoconstriction f o l l o w i n g n e r v e s t i m u l a t i o n a n d i n o n e , n o significant vasomotor response was elicited after aadrenoceptor blockade.
I~
CBF
Response stability and autonomic blockade Once established, cutaneous vasodilator responses were quite stable when evoked repeate d l y o v e r t i m e (Fig. 4). E v o k e d s u d o m o t o r a n d
CBF
0 0.5 HZ
16 Hz
__~ 1 HZ
16 Hz
2 Hz
4 Hz
16 Hz ~, 6 Hexamethonium (20 mg/kg)
8 Hz
16 Hz
Fig. 3. Continuation of cutaneous blood flow (CBF) responses from Fig. 2 (after prazosin and yohimbine). The intensity of stimulation was increased from 6 to 9 V, which in turn, unmasked a residual vasodilator response. Note that, as the frequency of stimulation increased, mixed vasoconstrictor and vasodilator responses are manifest. Administration of the ganglionic blocking drug, hexamethonium (20 mg/kg i.v.), totally abolished all responses to preganglionic nerve stimulation.
44
"J~
EDR
mal responses. A higher dose of C 6 (20 m g / k g i.v.; data not shown) totally eliminated all nerve evoked responses.
(mY) i
Discussion
2
0
251
I l
(U ~) 20 15 i
0
Prop Atropine
C~
Fig. 5. Composite representation of electrodermal (EDR) and cutaneous vasodilator (CBF) responses due to preganglionic sympathetic nerve stimulation before and after treatment with autonomic blocking agents in anesthetized cats. Solid bars, control responses to sympathetic nerve stimulation. Crosshatched bars, magnitude of responses 10 to 15 rain after i.v. administration of propranolol (prop; 1 m g / k g ) , atropine (1 m g / k g ) or hexametbonium (C6; 1.5 m g / k g ) . The vasodilator responses are expressed in arbitrary units (U 2) which represent area under the vasodilator curve. For the propranolol group, three cats were depleted of monoamines with reserpine and a-n,ethyl-p-tyrosine and one was pretreated with yohimbine and prazosin. In the experiments using atropine, one cat was depleted of monoamines and two were pretreated with yohimbine and prazosin. For hexamethonium, two cats were m o n o a m i n e depleted, two were pretreated with guanethidine and two were a-adrenoceptor blocked. Values are m e a n s + S E M . * * P < 0.01. Stimulation parameters: 6 to 10 V: I - 2 ms pulse duration: 7-10-s trains presented at 2- to 3-min intervals.
vasodilator responses were observed before and after blockade with i.v. propranolol (1 mg/kg), atropine (1 m g / k g ) and two different dosages (1.5 and 20 mg/kg) of hexamethonium (C6). As shown (Figs. 4 and 5) neither /3-adrenergic nor muscarinic receptor blockade altered neurally evoked cutaneous vasodilation. The sudomotor (EDR) system was almost totally blocked by atropine but not affected by propranolol. With regard to ganglionic blockade, the low dose of C 6 (1.5 m g / k g i.v.) almost completely eliminated the vasodilator responses while having no significant action on the simultaneously evoked electroder-
In the present study, preganglionic sympathetic nerve evoked responses (forepaw cutaneous blood flow alterations as measured by Laser Doppler flowmetry and neurally elicited sudomotot responses) were studied in chloralose anesthetized cats. In non-pretreated animals, nerve stimulation produced near maximal sympatheticcholinergic electrodermal responses and frequency-related cutaneous vasoconstrictions. The observation that vasoconstrictor responses were selectively abolished by combined a~- and c~2adrenoceptor antagonism (with the predominant effect being a2-adrenoceptor mediated) is in total agreement with our previous reports [14,15]. Others also have recently demonstrated a predominance of innervated posqunctional a2-adrenoceptors in small terminal arterioles in vivo [19]. Most studies in this area have used dogs pretreated with guanethidine [1,2,6,20,22], although cats also have been studied [4,22]. In addition to guanethidine, reserpine and bretylium have been utilized for anti-adrenergic effects [1,20,22]. Cutaneous flow was measured indirectly by subtraction from total limb blood flow by skinning or occlusion of the paw [2,20,22], by direct measurement of perfusion pressure in small arteries [1,6], or by measurement of surface temperature changes in the footpad of the cats [4]. The overall consensus of these studies is that sympathetic nerve stimulation produces an active vasodilator response in cutaneous blood vessels of the paw by action on receptors that are non-cholinergic, non-adrenergic and non-histaminergic. There also is evidence that the cutaneous vasodilator response is not mediated by bradykinin [1,2], /3-adrenergic [1-3,18], or prostaglandin [2] mechanisms. Bell [2,3] believes that dopamine may be the vasodilator mediator; however, the lack of specificity of the antagonists used, along with the presence of active vasodilation following reserpine [1,18] or chronic guanethidine pretreat-
45 merit [4], makes this conclusion less than totally convincing. Recently, calcitonin gene-related peptide has been shown to produce a very large increase in skin blood flow in humans [10] and is, along with a variety of other peptides, a possible candidate for the vasodilatory substance. This question will likely not be resolved until sufficiently specific antagonists for the various putative vasodilators are developed. The present study in cats used a more direct approach (Laser Doppler flowmetry) and compared three different modes of eliminating the noradrenergic vasoconstrictor component. Cutaneous vasodilator responses were unmasked following either prejunctional (monoamine depletion or guanethidine) or postjunctional (aadrenoceptor) antagonism, although not all animals tested exhibited a pronounced vasodilator effect. Difficulty in obtaining consistent vasodilation in adrenergic blocked cats is consistent with the early report by Zimmerman [22] in which he observed non-cholinergic cutaneous vasodilation in less than half of the animals tested. The reason for this variability is not clear although in the present study, there was no apparent relationship with regard to the particular anti-adrenergic regimen used. Of the three dosage regimens used, guanethidine most clearly decreased basal cutaneous blood flow. A similar depressant increase in resistance to flow has been reported with this agent in birds [3,18]. It may be that continuous release of catecholamines is responsible for this effect [9] although a non-specific effect might be present as guanethidine also decreased electrodermal response amplitude. The contribution of this depressant action in generating subsequent vasodilation is unknown. It is of interest, however, that vasodilator responses are manifest after a-adrenoceptor blockade (which by itself did not depress basal skin blood flow). Residual cutaneous vasodilation due to sympathetic nerve stimulation appears to be a general phenomenon as active cutaneous vasodilation is observed after adrenergic neuron blockade in a variety of other species including rats [8], birds [3,18] and humans [11]. In some studies postganglionic nerves or dorsal roots were stimulated
with the potential of retrograde activation of sensory nerve fibers and subsequent release of vasoactive peptides such as substance P [4,8,12]. In the present investigation, as in many others [4,6,17,20,22], the decentralized preganglionic nerve trunk was stimulated. This, along with the sensitivity to hexamethonium, strongly supports activation of sympathetic vasodilator efferent nerve fibers. A high sensitivity to hexamethonium blockade has also been demonstrated for muscle vasoconstrictor responses [5], whereas neurally evoked cutaneous vasoconstrictor responses are resistant to blockade by low doses of hexamethonium [5,15]. It has been proposed that cutaneous vasodilation is not a distinct event, but is produced by vasodilator substances secondary to sudomotor glandular activation [7,16]. The observation that atropine abolishes the sudomotor responses but not the cutaneous vasodilation combined with the clear separation of these neurally evoked events by low doses of hexamethonium, argues against their hypothesis. In addition, active sympathetic evoked cutaneous vasodilation is seen in birds even though they arc devoid of a sudomotor system [3,18]. The present results are totally in agreement with those of Bell et al. [4] in which they performed similar experiments using surface temperature changes as an index of cutaneous blood flow. In their study using guanethidine pre-treated cats they found that atropine selectively blocked sudomotor activation, whereas low doses of hexamethonium selectively inhibited preganglionic evoked temperature elevations. In extending the work by Bell et al. [4], we have demonstrated that active non-cholinergic, non-adrenergic cutaneous vasodilation can be measured in the forepaws of anesthetized cats using Laser Doppler flowmetry. Both pre(guanethidine pretreatment and monoamine depletion) and postjunctional ( a - a d r e n o c e p t o r blockade) elimination of the vasoconstrictor component was effective in unmasking vasodilation although basal cutaneous blood flow is better preserved by a-adrenoceptor blockade. Finally, the selective blockade by atropine and low doses of hexamethonium argue against a causal linkage
46
between cutaneous vasodilation and sudomotor
activation.
Acknowledgements The authors thank Mrs. Linda Hess for her expert technical assistance and Ms. H. Whiteside for preparing the manuscript. We are grateful to KAO Corporation for financial assistance.
References 1 Ballard, D.R., Abboud, F.M, and Mayer, H.E., Release of a humoral vasodilator substance during neurogenic vasodilatation, Am. J. Physiol., 219 11970) 1451-1457. 2 Bell, C. and Lang, W.J., Evidence for dopaminergic w~sodilator innervation of the canine paw pad, Br. J. Pharmacol., 67 11979) 337-343. 3 Bell, C. and Rome, A.C., Pharmacological investigations of the vasodilator nerves supplying the duck's foot, Br. J. Pharmacol., 82 11984) 801-808. 4 Bell, C., J~inig, W., Kiimmel, H. and Xu, H., Differentiation of vasodilator and sudomotor responses in the cat paw pad to preganglionic sympathetic stimulation, J. Physiol., 364 (1985) 93-104. 5 Bell, C., Differentiation of neurogenic vasoconstrictor responses in skin and skeletal muscle of dog hindlimb, J. Auton. Nerv. Syst., 14 11985) 13-21. ~ Brody, M.J. and Shaffer, R.A., Distribution of vasodilator nerves in the canine hindlimb, Am. J. Physiol., 218 (19711) 470-474. 7 Fox, R.tt. and Hilton, S.M., Bradykinin formation in human skin as a factor in heat vasodilatation, J. Physiol., 142 (1958) 219-232. 8 Gamse, R. and Saria, A., Antidromic vasodilatation in the rat hindpaw measured by laser Doppler flowmetry: pharmacological modulation, J. Auton. Nerv. Syst., 19 (1987) 11)5-11 t. 9 Gillespie, J.S., The rat anococcygeus muscle and its response to nerve stimulation and to some drugs, Br. J. Pharmacol., 45 (1972) 4/)4-416.
10 Jiiger, K., Muench, R., Seifert, H., Beglinger, C., Bollinger, A. and Fischer, J.A., Calcitonin gene-related peptide (CGRP) causes redistribution of blood flow in humans, Eur. J. Clin. Pharmacol., 39 (19911) 401-494. I 1 Kellogg, D.L., Jr., Johnson, J,M. and Kosiba, W.A., Selective abolition of adrenergic vasoconstrictor responses in skin by local iontophoresis of bretylium, Am. J. Physiol., 257 (1989) H1599-HI61)6. 12 Khoshbaten, A. and Ferrell, W R . , Alterations in cat knee joint blood flow induced by electrical stimulation of articular afferents and efferents, J. Physiol., 430 (199/)) 77-86. 13 Koss. M.C. and Davison, M.A., Characteristics of lhe electrodermal response: A model for analysis of central sympathetic reactivity, Naunyn-Schmiedeberg's Arch. Pharmacol., 295 (1976) 153-158. 14 Koss, M.C., Characterization of adrenoceptor subtypes in cat cutaneous vasculature, J. Pharmacol. Exp. Ther.. 254 (1990) 221-227. 15 Koss, M.C., Kawarai, M. and lto, T., Neural activation of alpha-2 adrenoceptors in cat cutaneous vasculature, J. Pharmacol. Exp. Ther., 256 (1991) 1126 1131. 1~'~ Love, A.H.G. and Shanks, R.G., The relationship between the onset of sweating and vasodilatation in the forearm during body heating, J. Physiol., 162 11962) 121- 128. 17 l,undberg, J., Norgren, L., Ribbe, E., Ros~n, I., Steen, S., Th6rne J. and Wallin, 13.G., Direct evidence of active sympathetic vasodilatation in the skin of the h u m a n fool, J. Physiol.. 417 (1989) 437-446. 18 McGregor, D.D., Noncholinergic vasodilator innervation "3 in the feet of the ducks and chickens, Am. ,1. Physiol..,37 11979) Hl12-11117. I t) Ohyanagi, M., Faber, J.E. and Nishigaki, K., Differential activation of cry- and ~e2-adrenoceptors on microvascular smooth muscle during sympathetic nerve stimulation, Circ. Res., 68 (1991) 232-244. 2(1 Rolewicz, T.F. and Zimmerman, B.('., Peripheral distribution of cutaneous sympathetic vasodilator system, Am. J. Physiol., 223 (1972) 939-943. 21 Shepherd, A.P. and Oberg, P.A., Laser-Doppler blood l]owmetry, Kluwer Academic Publishers, Boston, 1990. 22 Zimmerman, B.G.. Comparison of sympathetic vasodilator innervation of hindlimb of the dog and cat, Am. J. Physiol.. 214 11968) 62-66.