Bulbospinal substance P and sympathetic regulation of the cardiovascular system: A review

Peptides, Vol. 6, Suppl. 2, pp. 69-74, 1985. '~ AnkhoInternationalInc. Printedin the U.S.A.

0196-9781/85$3.00 + .00

Bulbospinal Substance P and Sympathetic Regulation of the Cardiovascular System: A Review I C I N D A J. H E L K E , C L I V E L G. C H A R L T O N

A N D J I L L R. K E E L E R

Department of Pharmacology, Unifi~rmed Services University of the Health Scienees 4301 Jones Bridge Rd., Bethesda, MD 20814

HELKE, C. J., C. G. CHARLTON AND J. R. KEELER. Bulbospinal substance P and sympathetic regulation of the cardiovascular system: A review. PEPT1DES 6: Suppl. 2. 6%74, 1985.--The neurotransmitter role of substance P in mediating sympathoexcitatory effects in the spinal cord and cardiovascular effects elicited from the ventral medulla is presented. SP neurons located in the ventral medulla project to the intermediolateral cell column (IML) of the thoracic spinal cord. Intrathecal administration of a SP analog excites sympathetic outflow to the cardiovascular system. Likewise, activation of the ventral medulla results in sympathetically mediated increases in blood pressure and heart rate which are blocked with SP antagonists. The IML contained a high density of SP binding sites through which the peptide likely exerts its sympathoexcitatory influence on the cardiovascular system. Substance P Ventral medulla Intermediolateral cell column Substance P antagonists Substance P agonists Cardiovascular regulation Nucleus paragigantocellularis lateralis Substance P binding sites

Spinal cord

area over the trapezoid body (area M) [44]; a caudal area where the hypoglossal rootlets emerge from the medulla (area L) [38]; and an intermediate area between areas L and M (area S) [51] (Fig, 1). The most profound cardiovascular effects were elicited from the intermediate area [18, 20, 33, 61]. The nuclear structure most closely correlated with the cardiovascular sensitive portion of the ventral medulla is the lateral paragigantocellular nucleus [2] (Fig. 1). The intermediate region (area S) also overlays portions of the lateral extensions of the serotonergic neurons of the raphe magnus (B3), the nucleus reticularis gigantocellularis, pars ~, and the CI epinephrine containing cell group [49] (Fig. IA). Direct projections from the ventral medulla to the intermediolateral cell column (IML) of the spinal cord were shown with retrograde and anterograde anatomical tracers, and electrophysiologic techniques [1,7, 42, 47, 49]. Because the IML is the site of origin of sympathetic preganglionic neurons [1 !, 14, 30, 52], this descending medullary projection may therefore synapse with the preganglionic neurons and mediate the sympathetic responses elicited from the ventral medulla (Fig. IB). Several putative neurotransmitter systems are present in the ventral medulla and may mediate bulbospinal input to the sympathetic neurons which influence cardiovascular function. Serotonin [31,40], epinephrine [48,49], thyrotropin releasing hormone [3,28], and substance P are examples. Evidence is presented here to support the neurotransmitter role of substance P in bulbospinal pathways which mediate the sympathoexcitatory cardiovascular responses elicited from the ventral medulla.

THE ventral medulla oblongata is the focus of recent attention as a brain region important in the CNS regulation of cardiovascular function. The chemical neuroanatomy of the pathways connecting the ventral medulla with sites of autonomic outflow to the cardiovascular system is also a topic of active research. This review will present evidence that the neuropeptide, substance P, is a neurotransmitter in bulbospinal pathways which may mediate the sympathoexcitatory cardiovascular responses elicited from the ventral medulla. Dittmar [15,16] was the first to report the existence of a "vasomotor center" in the ventral medulla. More recently, Feldberg and Guertzenstein [18, 19, 20, 21,23] and others [4, 33, 43, 56, 58, 59, 61] showed that the ventral medulla was sensitive to a variety of pharmacologic agents (glycine, GABA, glutamate, bicuculline, pentobarbital, kainic acid, etc.) which produced alterations in cardiovascular function. The cardiovascular effects were largely mediated by the sympathetic nervous system [13, 33, 43]. That a specific site in the ventral medulla plays an important role in influencing central sympathetic pathways was further demonstrated during stimulation and lesion experiments. Electrical or excitatory amino acid-induced activation of the ventral medulla increased blood pressure and sympathetic nerve activity [12, 13, 39, 43, 49, 58]. Conversely, lesions decreased blood pressure to levels observed in spinal cord transected animals [12, 24, 43]. These studies indicated that an area in the ventral medulla is important in the maintenance of sympathetic vasomotor tone. Based on cardiovascular and ventilatory responses to medullary application of drugs, three distinct areas of the ventral surface of the medulla were defined (Fig. 1): a rostral

1q-his paper was presented as part of a symposium entitled "'Neuropeptides and central cardiovascular control."

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H E L K E , CHARLTON AND K E E L E R N E U R O A N A T O M I C A N D N E U R O C H E M I C A L E V I D E N C E FOR SUBSTANCE P

SP-immunoreactive cell bodies are present in the ventral medulla, where they are located in the midline raphe nuclei and more lateral in the nucleus paragigantocellularis lateralis and the nucleus interfascicularis hypoglossi [5, 29, 32, 37] (Figs. 2A and 2B). SP cell bodies are also present in a more lateral position ventral to the gigantocellular reticular nucleus [37]. Some of these SP-containing neurons also contain serotonin and/or thyrotropin-releasing hormone [8, 29, 32]. Nerve terminals containing SP are present in the IML of the thoracic spinal cord [30,37] (Figs. 2C and 2D), and there is an anatomic relationship between SP immunoreactive terminals and sympathetic preganglionic neurons [30]. That the IML SP innervation originated in suprfispinal areas was suggested by the finding that spinal cord hemisection caused a bilateral decrease in SP fibers in the IML [30]. We studied the ventral medullary SP neurons to determine whether they project to the IML. To do this we destroyed various hindbrain areas with electrolytic lesions and measured the SP content of microdissected areas of the thoracic spinal cord [26]. Unilateral lesions of the ventral medulla which included the nucleus interfascicularis hypoglossi decreased the SP content of the bilateral IML by 38%,. We have recently confirmed these data and also found a smaller (31%) unilateral reduction of SP in the IML in animals with nucleus paragigantocellularis lateralis lesions (Helke et al., unpublished observations). More rostral lesions, lesions of the midline raphe (obscurus and pallidus), or midbrain hemisections failed to alter the SP content of the IML [26]. These data suggested that SP in the IML originates in ventral medullary neurons. In addition to the specific anatomic localization of SP in neuronal pathways, another criterion for a neurotransmitter status for the peptide is the presence and specific anatomical localization of receptors. The kinetics and pharmacology of 125I-Bolton-Hunter labeled SP binding sites were recently described in spinal cord homogenates [9]. The binding of this ligand in the spinal cord had the following characteristics: high affinity; time, temperature and membrane concentration dependent; reversible; and was saturable [9]. Takano and Loewy [53] reported a high affinity binding site in the IML region using aH-SP incubated with slide mounted sections and quantitated by liquid scintillation counting. We have also used light microscopic autoradiography and computerized densitometric analysis to map the distribution and to characterize SP receptors in the spinal cord [10]. Very distinct high density binding was visualized in the IML (Fig. 3A), in the intercalated nucleus which extends between the IML and the central canal, and the central autonomic nucleus. Each of these nuclei contains sympathetic preganglionic neurons [l l, 14, 52]. These binding sites correlated well with the distribution of SP-immunoreactive nerve terminals (Fig. 2D) and with neurons in the IML as identified by cholinesterase stain (Fig. 3B). Longitudinal sections of the cord show that the high density of binding in the IML is non-continuous and irregular in dimension and again correlates with the cholinesterase-stained cells in the nucleus (Fig. 3C, 3D). This is due to the binding of SP to the noncontinuous cellular aggregations of the IML and the nucleus intercalatus. More direct evidence that the SP receptors in the IML are located on sympathetic preganglionic neurons per se was provided in suicide transport studies with the toxic lectin,

A.

B.

Vl

4--

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FIG. I. (A) Schematic of the ventral surface of the medulla and underlying structures. Left side: lateral paragigantocellular nucleus (bold outline), A5 cell group (vertical lines). C I cell group (dotted), lateral extension of B3 cell group (cross-hatched), AI cell group (horizontal lines). Right side: surface zones (M,S,L) defined by cardiovascular and ventilatory studies. (B) Schematic of coronal section of the medulla at the level of the arrow in A and of a neuronal connection to the IML. Abbreviations: IML--intermediolateral cell column; L~-area L (Loeschcke et al. [38]); M--area M (Mitchell et al. [44]); MLF--medial longitudinal fasciculus; nts--nucleus of the solitary tract; ntV~spinal trigeminal nucleus; nVII--facial nucleus; P--pyramidal tract; pgcl--lateral paragigantocellular nucleus; rga--nucleus reticularis gigantocellularis, pars a; rgi--nucleus reticularis gigantocellularis; rm--raphe magnus; S---area S (Schlafke et al. [51 ]); Tr--trapezoid body; V l--sixth (abducens) cranial nerve; Xll--twelfth (hypoglossal) cranial nerve.

ricin [27]. When injected into peripheral nerves, ricin is retrogradely transported to the cell body and causes cell death [57]. The resulting lesion is limited to neurons projecting to the injection site [57]. Ricin injections into the unilateral superior cervical ganglion in rats caused a unilateral loss of cholinesterase-stained cell bodies and of SP binding sites in the IML of the upper thoracic sections (primarily seen in T1-T2) [27]. These studies provided evidence for the cellular localization of SP binding sites on preganglionic sympathetic neurons. Depolarization-induced release of SP from the spinal cord was shown in hemisected neonatal spinal cords in v # r o [46], and from superfused spinal cords in vivo [60]. Release of SP specifically from the IML has not been directly demonstrated either in vivo or in v # r o . However, the in vivo efflux of endogenous SP into spinal cord superfusates was evoked by injections of neuroexcitatory doses of kainic acid into the ventral medulla [54]. Kainic acid resulted in a sympathetically mediated increase in blood pressure. Concomitant with the rise in blood pressure was a twofold increase in the amount of SP in the superfusion samples [54]. Whether this effiux of spinal cord SP is from the IML or the ventral horn which also receives projections from the ventral medulla [26], and whether it mediates the sympathetic excitation remain to be determined. F U N C T I O N A L E V I D E N C E FOR S U B S T A N C E P

lontophoresis of SP into the IML increased the firing rate of identified preganglionic neurons [3,22], which indicate

S U B S T A N C E P AND C A R D I O V A S C U L A R SYSTEM

71

FIG. 2. (A) SP-immunoreactive cell bodies in the rostral portion of the nucleus interfascicularis hypoglossi, i.e., lateral to the pyramidal tracts, ventrolateral to the inferior olive and near the ventral surface of the medulla. Indirect (FITC) immunocytochemistry was performed on colchicine-treated rat brain sections with a monoclonal antibody (Pel Freez) to SP. Bar=50/zm. (B) SP-immunoreactive cell bodies in the nucleus paragigantocellularis lateralis at the level of the caudal raphe magnus. Cell bodies are dorsal and lateral to the pyramidal tracts (P). Avidin-biotin complex (ABC) immunocytochemistry was performed on colchicine-treated rat brain sections. Bar=50/zm. (C) SP-immunoreactive nerve terminals in the intermediolateral cell column of the thoracic spinal cord. FITC-indirect immunocytochemistry as in (A). Bar=50/xm. (D) SP-immunoreactive nerve terminals in the intermediolateral cell column (IML), nucleus intercalatus and lamina X of the thoracic spinal cord. ABC immunocytochemistry as in (B). Bar 50/~m.

that SP may mediate excitatory sympathetic influences. This finding, in addition to our evidence for a ventral medulla to IML SP projection, and the important role of the ventral medulla in cardiovascular regulation, stimulated our interest in investigating the role of bulbospinal SP in mediating sympathoexcitatory input to the cardiovascular system. SP receptor agonists and antagonists were the primary tools used in these studies. Loewy and Sawyer [41] showed that a putative SP receptor antagonist ([D-Pro e, D-Trpr'TSP), administered intrathecally (IT) to anesthetized rats, caused a profound reduction in blood pressure. The SP antagonist also prevented the pressor responses characteristically elicited by kainic acid at the ventral medulla [41]. To more convincingly demonstrate the involvement of SP receptors, we chose to study the cardiovascular effects of intrathecal administration of four putative SP receptor antagonists. The antagonists studied were: (I) [D-Pro ~, D-TrpZ.']-SP; (II) [DPro ~, D-Phe 7, D-Trp"~]-SP; (III) [D-Arg ~, D-Pro ~, D-Trp 7~', Leu~q-SP; (IV) [D-Pro 4, D-Trpr.:"{}]-SP (4-11). We investigated their ability to interact with spinal cord SP receptors and to alter cardiovascular function. In rat spinal cord membrane preparation, these agents displaced ~ I - B H - S P binding, but were four orders of magnitude less potent than SP [9]. The IC50s were 5.0, 7.5, 7.0 and 45/xM for antagonists I-IV, respectively, compared to an IC50 of 0.46 nM for SP. In the initial in vivo studies. 50/zg of each antagonist was

administered intrathecally (IT). Antagonists I-III caused long lasting decreases (approximately 30 mmHg) in mean blood pressure whereas antagonist IV did not alter blood pressure [34]. To further assess whether a bulbospinal SP system is involved in mediating cardiovascular responses elicited from the ventral medulla, the GABA receptor antagonist, bicuculline, was applied to the ventral medulla to activate bulbospinal sympathetic pathways. When applied to a specific site in the intermediate area of the ventral medulla, bicuculline causes sympathetic mediated hypertension (+ 35-45 mmHg) and tachycardia (+70--90 bpm) [33,34]. After IT administration of each of the SP antagonists except IV, the normal bicuculline-induced increases in blood pressure and heart rate were blocked (Fig. 4) [34]. That intravenous administration of the same doses of the SP antagonists did not block the bicuculline-induced cardiovascular effects showed that the effects of IT administered antagonists were due to a site of action in the spinal cord and not due to peripheral leakage. The reversibility of the effects of antagonist III was shown by demonstrating that the bicuculline-induced responses returned within 1-2 hr following the IT administration of 5/xg (3.3 nmol) of the antagonist [34]. These data support the concept that spinal cord SP, presumably SP localized in the IML, is excitatory to the cardiovascular system. However, IT administration of drugs

72

H E L K E , CHARLTON AND K E E L E R

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FIG. 3. (A and B) Brightfield photomicrographs of autoradiograms of te~I-BH-SP binding sites in thoracic spinal cord and (C and D) matched cholinesterase-stained slices. A and C are transverse and B and D are longitudinal sections through the intermediolateral cell column (IML). Reprinted with permission from Charlton and Helke [10].

does not provide site selectivity in the spinal cord. The possibility that antagonist-induced blockade of the effects of SP primary afferent terminals in the dorsal horn contributed to the cardiovascular effects was ruled out in studies in which rats were pretreated with capsaicin. Neonatal administration of capsaicin destroys primary sensory neurons including those SP-containing afferents which terminate in the dorsal horn [45]. Capsaicin reduced the SP content of the dorsal horn but not the SP content of the IML [34]. Furthermore, neither the bicuculline-induced hypertension and tachycardia, nor the SP antagonist-induced blockade of the bicuculline responses was altered in capsaicin treated rats [34]. The sympathoexcitatory role for spinal cord SP was further verified by (1) analyzing the cardiovascular responses to spinal cord administration of a SP receptor agonist and (2) providing additional evidence that SP antagonists exert their effects through SP receptors by reversing the cardiovascular effects with the similar administration o f a SP receptor agonist. Intrathecal administration of SP (6 fmol to 60 nmol) was evaluated in preliminary experiments. However, we were unable to obtain dose-dependent pressor effects when SP was administered IT. Doses (>6 pmol) high enough to evoke cardiovascular responses when given IT produced similar effects when given IV and were consistent with those previously reported [6] following IV administration (Keeler and Helke, unpublished). These re-

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FIG. 4. Effect of a substance P receptor antagonist on the cardiovascular responses to bicuculline methiodide (BMI; 0.59 nmol) applied to the intermediate area (area S; see Fig. 1A) of the ventral surface of the medulla. (A) BMI responses 20 min after IT injection of 15 ~tl vehicle. (B) BMI responses 20 min after IT injection of [D-Arg], D-Pro~, D-Trp 7', Leu~q-SP (50/xg). Reprinted with permission from Keeler and Helke [34].

suits suggest that with IT doses of less than 6 pmol, SP was enzymatically inactivated before reaching its spinal cord site of action and at doses greater than 6 pmol, SP was leaking into the periphery to cause its effects. Therefore, a recently developed SP receptor agonist, [pGlu:', MePhe ~, MeGly+']SP(5-11) (DiME-SP), which is resistant to enzymatic degradation [36,50] was used. DiME-SP had been tested in peripheral bioassay systems [50], in CNS behavioral tests [ 17], and in brain tissue binding studies [25] and appeared to be an appropriate and selective SP receptor agonist. In preliminary studies, the ability of DiME-SP to interact with spinal cord SP receptors was demonstrated in binding studies. DiME-SP inhibited the saturable high affinity binding of r'~I-BH-SP to spinal cord membranes in a dose dependent manner with an IC50 of 1.5 /xM [35]. Intrathecal

SUBSTANCE

P AND CARDIOVASCULAR

SYSTEM

injections of DiME-SP (0.1-33 nmol) caused dose-related pressor and tachycardic responses [35]. Intravenous injections of the same doses of DiME-SP did not cause significant changes in blood pressure or heart, rate when evaluated at corresponding times following injection [35]. Pharmacologic blockade studies and measurement of plasma catecholam i n e s s h o w e d t h a t t h e c a r d i o v a s c u l a r effects r e s u l t i n g f r o m IT a d m i n i s t r a t i o n o f D i M E - S P w e r e m e d i a t e d b y the s y m p a t h e t i c n e r v o u s s y s t e m [35]. In a d d i t i o n , t h e b l o c k a d e o f t h e B M I - i n d u c e d p r e s s o r r e s p o n s e b y SP a n t a g o n i s t III w a s prev e n t e d b y c o n c u r r e n t IT i n j e c t i o n o f D i M E - S P (33 n m o l IT) [35]. In s u m m a r y , the d a t a r e v i e w e d h e r e (1) d e m o n s t r a t e a n i m p o r t a n t role for spinal cord S P in c a r d i o v a s c u l a r r e g u l a t i o n a n d (2) s u g g e s t t h a t t h e e x c i t a t o r y c a r d i o v a s c u l a r effects e v o k e d b y the s t i m u l a t i o n o f cell b o d i e s in t h e v e n t r a l m e d u l l a are in large p a r t m e d i a t e d b y S P via a b u l b o s p i n a l S P p a t h w a y . T h e s e d a t a a n d the r e s u l t s o f f u r t h e r studies o f t h e

73

neurochemistry of bulbospinal pathways which convey information to the cardiovascular system will be of importance for understanding the normal cardiovascular regulation. Perhaps even more important, this knowledge will allow the rational design of therapeutic agents with which to manipulate bulbospinal systems and alleviate the alterations which either cause or exacerbate a variety of cardiovascular disorders. In fact, r e c e n t e v i d e n c e suggests t h a t a b n o r m a l S P m e c h a n i s m s in t h e I M L m a y b e r e l a t e d to t h e p a t h o g e n e s i s o f h y p e r t e n s i o n at least in t h e s p o n t a n e o u s l y h y p e r t e n s i v e rat [55].

ACKNOWLEDGEMENTS We thank Elaine T. Phillips for technical assistance and Dr. Michael Hirsch for his helpful comments regarding this manuscript. The work was supported by NIH grant No. NS19317.

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