Regional peripheral and CNS hemodynamic effects of intrathecal administration of a substance P receptor agonist

Regional peripheral and CNS hemodynamic effects of intrathecal administration of a substance P receptor agonist

Journal q! the .,1utonomic .Verc,m.s 5).vtem. 21 ( 1987 ) I - 7 H:,evier l JAN 110759 Research Papers Regional peripheral and CNS hemodynamic effe...

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Journal q! the .,1utonomic .Verc,m.s 5).vtem. 21 ( 1987 ) I - 7 H:,evier

l

JAN 110759

Research Papers

Regional peripheral and CNS hemodynamic effects of intrathecal administration of a substance P receptor agonist C.J. Hclke i, E.T. Phillips i and J.T. O'Neill " I)elmrtmenrs of ; Pharmacoh~gv and ?Pediatricv. Un(/brnwd ,~'ert'tc(,.s ( :nit'ers;(v o/the Ih'alth Scwn,'e.v. Bethe.sda. M D 21)814 ( ~ .S..,l. (Received 22 April 1987) t Revised version received 17 July 1987 (Accepted 27 .lulx 19871

Key words: lntermediolateral cell column: lntrathecal: [ p G l u S , M e P h e S , M e G l y ' ~ ] - s u b s t a n c e Substance P agonist" Peripheral resistance in rats" Regional blood flow in rats

P~ ~~:

Summary Regional ('NS and peripheral hemodynamic effect,,, of the intrathecal (i.t.) administration of a substance P receptor agoni,,,t. [ ,r,Glu~.Mel~heS,MeGl?, q l-substance t~. i i ([DiMe]-SP), '.,,ere studied it] anesthetized rats with the radioactive microsphere techniqt.e. h was previously shown that [DiMe]-SP caused a sympatheticall), mediated increase in mean arterial pressure ~M.:"d~) by an action within the spinal cord. In this study. [l)iMe]-SP i5 arid 33 nmol. i.t.) increased MAP. 1he 5 nmol dose increased resistance in cutaneous, renal, splanchnic, and adrenal vascular beds hut decreased resi,,,tancc, and increased blood flow in ~,olne skeletal muscle bed:,. Total peripheral resistance was unchanged. The 33 nmol do:,e increased resistance in each peripheral vascular bed analvzed and increased total peripheral resistance. Whereas each dose increased heart rate, stroke volume and cardiac output were t.nchvnged with the 5 nmol dose and were reduced with the 33 nmol dose. Neither dose of [DiMc]-SP significantly altered regional brain or spinal cord blood flows. These data show that the i.t. administration of the SP agonist. [DiMeI-SP. increased vascular tone to mosl peripheral vascular beds whereas the low dose caused a ,.asodilation of skeletal muscle. These effects are consistent with the notion of a dose-related activation of SP receptors in the spinal cord affecting sympathetic ot, tflov, to the adrenals and to the vasct, lature.

Introduction The intrathecal (i.t.) administration of a stable substance P (SP) receptor agonist,. [ p G l u ~, MePheS,MeGly'~] -substance P~-11 ([DiMe]-SP), increased mean arterial pressure and heart rate [13]. The effects of SP receptor activation were due to an action within the spinal cord which increased sympathetic outflow [13,18,26]. The pressor and tachvcardic effects of [DiMe]-SP were blocked by

(orrewondence: ('.J. I lelke. Department of Pharmacology, [..,SI,.:ItS, 430I J,me,, Bridge Rd.. Bethesda. M D 20814, (..S.A. 0165-1838,/',~7/."$03.50

the i.t. administration of an SP receptor antagonist [9]. Similar responses were noted with the i.t. administration of SP [17,27]. SP binding sites have been located on preganglionic sympathetic neurons in the intermediolateral cell column (IML) [2,8,15.22,23]. Thus, it is likely that the cardiovascular effects seen after i.t. administration of [DiMe]-SP were mediated via this site. Whether preganglionic sympathetic neurons innervating specific vascular beds or the heart are preferentially excited by SP receptor activation has not been studied. Differential effects on blood flow and vascular resistance in various peripheral vascular beds were previously noted with the i.t.

" 1987 t-lsevier Science Publishers B.V. I Biomedical Division)

administration of an SP receptor antagonist, [dArgl.D-ProZ.D-TrpT"'~,Leul~]-substance P [9]. The purpose of the studies reported here was to investigate the effects of the i.t. administration of the SP receptor agonist, [DiMe]-SP, on regional peripheral and CNS hemodynamics to assess whether the [DiMe]-SP-induced increase in blood pressure was due to a generalized peripheral vasoconstriction.

Materials and Methods

Animals Male Sprague-Dawley rats (220-285 g) were anesthetized with a-chloralose (Division Pharmacie, Clichy, France) and urethane (Sigma, St. Louis, MO) (60 and 800 m g / k g i.p.). The rats were artificially ventilated with room air using a Harvard rodent ventilator. Blood pH and gases were maintained within normal limits. A femoral artery was cannulated to measure blood pressure and a femoral vein was cannulated for administration of drugs. Mean arterial pressure (MAP), heart rate (HR), ECG, and rectal temperature were monitored. Data presented for MAP, HR, blood pH and gases correspond to the values at the time of the blood flow determination (i.e. radioactive microsphere administration). A cannula was inserted into the subarachnoid space with the tip at the 9-10th thoracic segment [12,25]. The placement of the intrathecal cannula was verified at necropsy.

Blood flow determination Regional CNS and peripheral tissue blood flows and vascular resistances, and cardiac output were determined with the radioactive microsphere technique (reference sampling method). For injection of microspheres, the left ventricle was catheterized via the right carotid arte~. The position of the catheter tip was evaluated by the presence of the left ventricular pressure pulse and was verified at autopsy. A femoral artery catheter was used for collection of the reference blood sample. Microspheres (15/~m), labelled with Cerium-141 (14ICe) and Strontium-85 (85Sr) (10 mCi/g, 3M Tracer Microspheres, 3M, St. Paul, MN) were

suspended in saline containing 1%, dextran and 0.005~ Twecn 80, sonicated and vortexed. Microspheres (300.000-400.000, 900 ~1) were in letted and flushed into the left ventricle tover 1 mini with blood from a donor rat. A reference sample was collected from the femoral artery at a rate of 0.616 m l / m i n with a Harvard infusion/withdrawal pump. The collection of the reference sampie commenced 10 s prior tt~ the microsphere injection and stopped 1 rnin aller its completion. Blood pressure was carefully monitored throughout the injection/withdrawal procedure, ~dcquate ventricular mixing t)l' tilt.' >pheres x~,as ,:t~sessed by comparing calculated flows in the right and left kidneys. Phosphate buffered saline (PBS) vehicle or the SP agonist, [DiMeI.-SP. was admimstered 8 -10 rain prior to the blood flow determm~,,tion. This time point was shown in previous stud-, ies [11] to be the time of the peak MAP response due to i.t. [DiMe]-SP. Each ral received i.t. rejections of vehicle and one dose of [I)iMe]-SP. Microspheres labeled with either ~a;('e or '~5S~ followed each i.t. injection and permitted the determination of hemodynamic events at two time points, with each rat serving as its own control. Microspheres labeled with ~41Ce and ~SSr were given alternately for the first and second hemodvnamic determinations. The rats were euthanized, tissues removed, blotted on gauze, and weighed. Reference blood samples, tissues, and standards were counted in a 'l-racor 1185 Gamma Counter. Counts were t:of retted for interaction betweet,, the two isotopc.~. Tissue blood flow and vascular resistance, total peripheral resistance and ca~-diac output were calculated as previously described by Hexmann [10] and Stanek et al. [21].

Drugs Solutions of [pGlu~.MePhe~.MeGly'~i-SR_.~ ([DiMe]-SP) (Peninsula Laboratories, Belmont. CA) were prepared fresh daily from dried aliquots. [DiMe]-SP was dissolved in phosphate buffered saline (PBS) and administered intrathecally in a volume of 7.5 /,tl followed by a 7.5 ~tl PBS flush. Previous studies showed that the doses administered i.t. exerted their effects through a CNS site of action [13].

Statistics Data are presented as mean + S.E.M. with significance level at P < 0.05. Data were analyzed by Student's t-test for paired data.

Results

Ef/ect of i.t. administration of [DiMe]-SP on peripheral hemodynamics Previous experiments showed that MAP, IIR, cardiac output, total peripheral resistance and tissue blood flows were reproducible with sequential administration of 14t(Te and SSSr labeled microspheres [9,11,17]. A 5 nmol i.t. dose of [DiMe]-SP caused a 19 mm Hg increase in MAP with no significant increase in total peripheral resistance (Table 1). This dose of [DiMe]-SP also elevated heart rate an avcrage of 63 bpm. The small increase in cardiac output (11 ml/min) and the reduction in stroke volume (12 p,l/beat) were not statistically significant. In contrast, the 33 nmol dose of [DiMe]-SP increased MAP (28 mm Hg), TPR (0.59 mm Hg/"rnl/min), and FIR (47 beats/min). Furthermore, the 33 nmol dose of [DiMe]-SP decreased cardiac output (31 ml/min) and stroke volume (82 /.tl/beat) (Table I). Blood pH and gases were analyzed immediately prior to each administration of microspheres and no significant differences were detected ('Fable I). Regional blood flow and vascular resistance data determined after an i.t. injection of vehicle and of 5 nmol dose of [DiMe]-SP are shown in

Table II and the corresponding data after administration of vehicle and 33 nmol dose of [DiMeJ-SP are shown in Tablc lIl. The i.t. administration of a 5 nmol dose of [DiMe]-SP increased vascular resistance in cutaneous (lumbar and abdominal), adrenal, renal, and portions of the splanchnic (stomach and small intestine) circulations (Table II). With the exception of the kidneys where the increasc in resistance was accompanied by a reduction in blood flow, blood flows were not decreased. In contrast, vascular resistance was decreased and blood flow was increased in abdominal muscle, but neither was significantly changed in triceps muscle (Table I11. The pattern of changes in vascular resistance and in tissue blood flows were not identical between the 5 and 33 nmol doses of [DiMe]-SP. In contrast to the lower dose. the higher dose caused an increase in skeletal muscle vascular resistance in both the triceps and abdominal muscles (Table 111). Vascular resistance was increased in all other peripheral vascular beds studied. The increases in resistance were accompanied by reductions in blood flow in the kidneys, and triceps muscle but not in cutaneous, adrenal or splanchnic circulations. No significant differences between right and left kidney blood flows were noted before or after either dose of [DiMe]-SP.

EfJ~'ct of i.t. administration of [Dime/-SP on brain and spinal cord hemodvnamic.s" Thc i.t. administration of 5 nmol [DiMe]-SP did not change blood flow or vascular resistance in any of the brain structures anal'¢zed (Table II).

"I'A BLt~ I

Islet'cot ~I [DiMe]-SP (i.t. j on hemodvnamic parameter.s', hlood ptt and ga.s'e~'

Mean arterial pressure ( m m Hgl Total peripheral resistance (ram H g / m l / m i n ) ('ardiacoutput{ml/min) I.'learl rate ( b e a t s , / m i n ) Stroke volume ( p . I / b e a t ) Blood p l l Blood p('O z Blood p O .

Control

5 nmol [Dr Me]-SP

('ontrol

121 1.22 102 447 234 7.39 36.3 77.9

140 _• 4 * 1.26 ± 0.08 113 _+ 6 510 8~' -.~ *. 222 +_ 18 7.40 :~ 0.01 36.0 ± 1.5 82.3 _+ 2.9

124 5. 11.97 ± 132 _478 . .+ 275 + 7.36 ± 39.6 ± 73.7 ~.

_+_ 3 ± 0.09 _+ 8 ±24 ± 31 .; 0.01 _... 2.4 ± 2.5

.

33 nmol [I)iMe]-SP 5 0.116 6 8 12 0.02 3.0 3.1

* P <:: 0.05 c o m p a r e d to c o r r e s p o n d i n g vehicle control data using Student's t-test for paired data. n = 5 6.

15_"~ +._ 8 * 1.56 _~ 0.18 * 101 1-11 * '~'~'.~ -~ I 1 * 193 +_20 * 7 . 3 2 - 0.02 38.1 ~ 3.0 76.5 ~ 3.0

IAI]LI!. II

Effect t!f [ D i M e ] - S I ~ (5 nmol i.t. i on reel,real hlood I b m and ,',,cular r e ~ . , m n , ' c in :a.'. ('trculauon

Ttssue ]~ow (toil'rain., 10()g) Control

Cutaneous Lumbar Abdominal Muscular Triceps Abdomimd Renal R. K i d n e y k. K i d n e ? Splanchnic Stomach Sin. I n t e s t i n e Liver Adrenals Brain Forebrain (..'erebellum Medulla/Pons Spinal Cord Cervical Thoracic Lumbosacral *

P < 0.05 c o m p a r e d

TABLI-

I , , , , ui~,," rt'xtvtancc (ram lift

/DtMe]..'¢t"

", o/ { , n t r . :

( ,,::, ..,,

, . . . . ~t,t

it~ ~ .,,~

..

l,..m.,-.

: I)i If,.: .', ,'

i(}.l + 12.7 --

1.5 2.5

7.8 :r c;.7 -.

I.I I ..',

"" "{,

13.6.1 I1..~t~

.2.31 2.51

.2( } i;'."

16.3 ~ 5.3 ~-

3.¢, t.).7

2(i.2 .:_ 7.5 -~

5.3 IJI

124 142 *

t~ 55 24.-;a

2.28 2.45

9.41 • 2..4'. 2!1.21 - 2.i.-.

~,, :-:2 *

4 1 7 . 2 t_ 4 2 . 6 388.5 :t 32.7

78 * 74 *

0...., 1:. 0 . 0 1 .2~ L t).(12

11.3~ .r: {}.O.a

1{,5 *

U ,if !. {..1~,;

]7,7 *

~1~ ~;,x 100 8g

',.47. 0.46 2.42 0i2

0.45 0.04 (}.27 11.(}1

4 . 7 3 .. ,I ..¢ o . 6 2 -- ~l.{i: 2.81 .r (}.).~ o.16 0.1.,-

158 * 135 * ! !6 125 "

537.1 ~ 37.4 5 2 6 . 8 2 29.7 38.7 272.3 52.8 1 082.6

~_ 5.6 * 23.3 ! 6.0 ± 200.7

3(}.9 240.3 52.7 956.8

.[ 2.6 +_ 27.~ 2 5.1 - 118.5

-1 ¸ .

2 ÷ ~

16&1

:. ,

...

.3~

.

: ." ~

"

" :~]

"

98.5 ± 148.2-!. 140.2 +

7.9 1(I.2 9.9

117.1 .t 149.5 ± 167.2 !.

g.6 10.8 13.8

i 19 1(}1 119

I.;1 r 11.12 0.,";3 ÷.. 0 . 4 6 0 . 8 8 -- 0 . 0 6

~,.~.'}' ~. {}.u'.~ I).96 ,-. 0A;7 0 . 8 6 -. t}.t,

,45. } i6 t~S

120.0 L 117.9 ± 114.8 ~-

10.8 9.7 14.0

13~.., " ~ _- 7.S 126.1 ~- 12.0 126.4 ~ 13.2

111 1l.}7 ll{~

!AI5 L O . 0 9 1.06 ~-{}.10 1.12 L. 0.12

l.t)'7 .. ¢).U: 1.15 -:. i} !;.. I.l{, j {}.1i

l l.,. w 1 N 11}4

to v e h i c l e c o n t r o l u s i n g S t u d e n t ' s

t-test f o r p a i r e d d a t a . n - t,.

Ill

l~[fi'ct o f [ D i M e / - S P {33 nrnol t.t.) on regtonal blood flow and cascular resistan{ e in ruts Circuhnion

"Iissue flow (roll" rain ..," 100 g) Control

Cutaneous I.umbar Abdominal Muscular Triceps Abdolnina[ Renal R. K i d n e y L. K i d n e y Splanchnic Stomach Sin. I n t e s t i n e Liver Adrenals Brain Forebrain Cerebellum Medulla/Pons Spinal cord Cervical Thoracic Lumbosacral *

P < 0.05 compared

l "ascular resistance (ram t l g , m l / rain / I00 ~¢.)

/DiMe]-SP

'~ of Control

C, mtrol

[DiMe]-SP

% of C'ontrol

I 4

72

If).6g +. 0 . 8 2

2.4

74

8.07 :- 1.49

19.68 .!: ~ 34 13.24 .~. 1.92

.84 * 164 *

~632- 1.17 lq.tM ~- 2.01

1 4 . 7 4 t ' :," 30.55 ! 4.),.':

222 * 153 *

I 1.9 _+ 17.4 ±

0.5 2.5

8.6 4 12.8 ~:

22. I ). 6.6 ~

4.6 ().9

11.2-4

16

51 *

5.2 I

1I

-79

572.8 ~ 574.4 ±

025 94.9

2 5 t . 6 .-. 23.2 2 4 6 . 4 4 32.9

56.0 + 6.1 3 9 7 . 8 42_ 88.6 61.7_+ 18.3 I 674.4 ± 205.9

44 * 43 *

0 . 2 ? 1 0.05 0.27 ~: 0.08

6 ~, _,_ tl JY: (1.66 t I}.0~

2,2. * 244 *

36.2 :!- 5.1 2 8 3 . 8 .+_ 91.1 4 4 . 3 - ~ 17.9 1 187.8 _+ 323.4

65 71 72 71

2.37 0.43 2.82 0.08

5.27 0.71 5.99 0.16

222 148 212 20t1

173.5 4: t7.5 2 4 8 . 6 ± 34.6 2 3 1 . 6 ± 28.2

172.2 ± 15.3 201.8± 18.4 2(18.4 -I. 24.3

99 81 9(1

0 . 7 6 ~- 0 . 0 8 ).55! 0.08 {).57 ~. 0.(16

(I.98 3: t).o,x, 0.79 t.{!.ll 11.77 :. I i i

128 144 *

132.7 _+_ 4 4 . 4 189.2 + 25.1 165.8 .t- 1 8 . 2

128.8 3:- 41.8 168.6 ± 19.8 138.6 -y 20.0

97 89 84

1.74 .f 0 . 6 6 0.71 ± 0 . 0 9 {).70 -t 0 . 0 9

2.24 ± 0 . 8 9 0 . 9 6 +- 0 . 1 4 1.17 :~: ILl,':,

129 135 148 *

to vehicle

contrc)l using Student's

t-test f o r p a i r e d d a t a . n : 5.

+ 4 L :.

0.29 0.15 0.71 0.01

• I..-.'} ±0.14 :; 1.76 ~ 0.1)3

* * * *

34 *

Likewise, these hen3odynamic parameters were not altered in any spinal cord level in [DiMe]-SPtreated rats. The higher dose (33 nmol) of [DiMelSP likewise was without effect on brain or spinal cord blood flow: however vascular resistance increased in the cerebellum, medulla/pons, and lumbosacral spinal cord (lable III).

Discussion

This study provides information on cardiovascular changes that occur with i.t. administration of an SP receptor agonist. Whereas previous studies showed blood pressure aherations induced by i.t. administration of [DiMe]-SP [13], this is the first report of regional hemodynamic effects of an SP receptor agonist. [DiMe]-SP, the SP receptor agonist uscd in the current study and in our previous studies [9,131 is a metabolically stable SP analog developed and characterized by lverseq and colleagues [3,19]. Because it is resistant to metabolic degradation, uncertainties about potential biological activity of degradative fragments of SP (eg. SP I_ ~) [6.161 arc largely eliminated. The baseline regional blood flow and cardiac output data obtained in this study are in agrccment with previous studies of peripheral and central hemodynamics in the rat [7,9,17,21,24]. Methodoh)gic issues relatcd to our use of microsphercs to assess regional blood flows in the rat were discussed previously [9]. Whereas both doses of [DiMe]-SP (5 and 33 nmol, i.t.) increased MAP and heart rate as previously reported [13], the vascular effects of these doses were not identical. The lower dose of [I,)iMe]-SP had variable effects on regional peripheral vascular resistances and no significant effect on total peripheral resistance. The vasodilation in certain skeletal muscle beds wax apparently countered by vasoconstriction in the cutaneous and mesenteric circulations. This pattern of hemodxnamic changes is consistent with the idea of elevated circulating levels of epinephrine causing fl receptor-mediated dilation in muscles and ~ rcceptor-mediated constriction elsewhere. The higher (33 nmol) dose of [DiMe]-SP significantly increased total peripheral resistance and MAP by

causing a vasoconstriction in each peripheral vascular bed analyzed, including skeletal muscles. Despite the elevated MAP, the vasoconstriction was great enough to cause significant reductions in blood flow in certain regions (triceps. kidneys). The reduced cardiac output and stroke volume resulting from the 33 nmol dose of [DiMe]-SP was liken due to the elevated afterload, although the contribution of reduced venous return due to possible hepatic venoconstriction can not be ruled out. The generalized vasoconstriction seen in response to the higher (33 nmol) dose of [DiMe]-SP is consistent with increased discharge of s,,mpathetic neurons to the cardiovascular system and the subsequent release of norepincphrine. Whereas it was previously. shown that high doses of [I)iMe]-SP (33 nmol i.t.) increased both epinephrine and norepinephrine [13]. direct evidence that plasma epinephrine but not norepinephrine is elevated by the low doses of SP of [I)iMe]-SP ix n o t available. Neither dose of [DiMe]-SP administered i.t. altered spinal cord or cerebral blood flow. The vasoconstriction seen in certain CNS regions (cerebellum, medulla/pons, lumbosacral cord) following the high (33 nmol) dose of [DiMe]-SP was likely an autoregulatory response to maintain a constant levcl of cerebral blood flow under conditions of elevated MAP. These data contrast wi*.h a local non-specific effect on the SP receptor antagonist. [D-A rg l,I~-ProZ.D--l-rp~ '~.Leu 11]-SP, which upon i.t. administration significantly reduced spinal cord blood flow in the region of the injection [9]. The ability of intrathecallv administered [DiMe]-SP to increase MAP, and incrcase peripheral vascular resistance in all vascular beds analyzed suggests that SP receptor activation could cxcitc most. if not all, sympathetic neurons which influence vasomotor tone. However. our previous data with the SP receptor antagonist. [])-Arg ~. D-Pro -~, D-'FrpV"~.LeuJl]-SP suggested that tonically active SP-containing neurons were primarily involved in sympathetic outflow to the venous vasculature [9]. Thus. tonic vasomotor input from SP-containing neurons to sympathetic preganglionic neurons influenced only a portion of the spinal cord SP binding sites whereas a larger

population of receptors could be activated by i.t. administration of an SP receptor agonist or bx phasic activation of bulbospinal sympathoexcitatory systems in the ventral medulla [12-- 14]. Previous work showed that the intrathecal administration of [DiMe]-SP increased MAP and HR due an action within the spinal cord [13]. In addition, functional data demonstrating a [DiMe]SP-induced increase in sympathetic outflow and knowledge of the presence of excitatory SP receptors on preganglionic sympathetic neurons in the IML [1,5,8] support the notion that the effects of i.t. administration of [DiMe]-SP were due to effects in the IML. However, SP binding sites are also present in the dorsal and ventral horns of the spinal cord and a sympathoexcitatory effect of [DiMe]-SP acting through either of these system,~ cannot be ruled out. Activation of at supraspinal reflex by the i.t. administration of [DiMe]-SP is unlikely because spinal transaction does not block the [DiMe]-SP-induced pressor effects (unpublished observations). Because primary afferent dorsal horn SP pathways are involved in sensory modalities, theoretically, they could be involved in known somatosympathetic and viscerosympathetic spinal cardiovascular reflexes [4.20]. However, because of the demonstrated excitatory effects of SP receptor activation on preganglionic sympathetic neurons [1,5], the primary sympathoexcitatory site of action is likely to be the IM[,,

Acknowledgements We thank Martha McShane and the Department of Physiology, Uniformed Services University of the Health Sciences for use of their blood gas analyzer. This work was supported by NIH Grants R01-NS24876 (to C.J.H.) and R23HD20301 (to J.T.O'N.).

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