Stimulation of Serotonin2 Receptors in the Ventrolateral Medulla of the Cat Results in Nonuniform Increases in Sympathetic Outflow

 
Stimulation of Serotonin2 Receptors in the Ventrolateral Medulla of the Cat Results in Nonuniform Increases in Sympathetic Outflow
1267

            Stimulation of Serotonin2 Receptors in the
           Ventrolateral Medulla of the Cat Results in
          Nonuniform Increases in Sympathetic Outflow
                  Aloke K. Mandal, Kenneth J. Kellar, Wesley P. Norman, and Richard A. Gillis

         Topical application of the serotonin2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane
         or DOI, in a dose of 30 jig/side to the intermediate area of the ventrolateral surface of the
         medulla produced a significant increase in mean arterial pressure with no significant change
         in heart rate both in intact animals (n=8) and in cervically vagotomized animals (n=3). The
         pressor response of DOI was blocked by pretreatment of the intermediate area with ketanserin,
         a serotonin2 antagonist (n =7). Pretreatment with intravenous phentolamine did not block the
         pressor response of DOI (n=3). However, this pressor response could be counteracted by
         intravenous propranolol (n=5) or by bilateral stellate ganglionectomy (n=3). These data
         suggest that sympathoexcitation by centrally applied DOI selectively increased cardiac inotropy
         but not chronotropy. Further studies indicate that DOI increased contractile force without
         increasing heart rate and that the positive inotropic effect of DOI could be counteracted by
         bilateral stellate ganglionectomy. Bilateral microinjections of DOI into the subretrofacial
         nucleus in a dose of 100 ng (n=3) and a dose of 300 ng (n=3) increased mean arterial blood
         pressure by 23±2 and 44±6 mm Hg, respectively, without producing any changes in heart rate.
         These data suggest that DOI has a central site of action in the ventrolateral medulla,
         presumably at the subretrofacial nucleus, which leads to selective sympathoexcitation of the
         cardiac ventricles. (Circulation Research 1990;67:1267-1280)

W e recently reported evidence that sug-                           receptors.12 At the time of these studies, McCall et
            gested that serotonin (5 -HT) applied top-             al3,4 reported that intravenous administration of
            ically to the intermediate area of the                 5-HT2 agonist drugs raised blood pressure and in-
ventrolateral medulla of the cat could stimulate                   creased central sympathetic outflow. These data sug-
serotonin2 (5-HT2) receptors, resulting in a rise in               gested to us that within the central nervous system
arterial blood pressure.' Indeed, our evidence for                 5-HT simultaneously activates 5-HTlA receptors,
making such a statement was very indirect and con-                 which lower blood pressure, and 5-HT2 receptors,
sisted of demonstrating that blockade of 5-HT2 re-                 which increase blood pressure.
ceptors at the intermediate area with topical appli-                  In testing the statement that activation of central
cation of the 5 -HT2 receptor antagonist drug                      nervous system 5-HT2 receptors causes an increase in
ketanserin, at the same site, augmented the hypoten-               sympathetic outflow, resulting in an increase in blood
sive effect of 5 -HT. Presumably, the hypotensive                  pressure, we first sought to determine whether activa-
effect of 5 -HT was a result of activation of 5 -HT1A              tion of 5 -HT2 receptors at the level of the intermediate
                                                                   area of the ventrolateral surface of the medulla would
   From the Departments of Pharmacology (R.A.G., K.J.K.,           cause an increase in blood pressure. We chose to look
A.K.M.) and Anatomy (W.P.N.), Georgetown University School
of Medicine, Washington, D.C.                                      at this area initially because it has been shown that
   A portion of this work was presented at the 19th Annual         many drugs that have a central action on cardiovascular
Meeting of the Society for Neuroscience, October 29-November 3,    activity and sympathetic outflow often act at the inter-
1989, Phoenix, Arizona.                                            mediate area.5,6 To this end, we evaluated the effects of
   Supported by a grant to R.A.G. from Byk-Gulden Pharmazeu-
tica, Konstanz, FRG. A.K.M. is a recipient of a Medical Student    topical application to the intermediate area of the
Research Fellowship in Pharnacology-Clinical Pharmacology          selective 5-HT2 agonist agent 1-(2,5-dimethoxy-4-io-
from the Pharmaceutical Manufacturers Association Foundation,      dophenyl)-2-aminopropane, commonly referred to as
Washington, D.C.                                                   DOI. This compound has been shown to selectively
   Address for correspondence: Richard A. Gillis, PhD, Depart-     label 5-HT2 receptor binding sites and has been pro-
ment of Pharmacology, Georgetown University School of Medi-
cine, 3900 Reservoir Rd., NW, Washington, DC 20007.                posed as a selective 5-HT2 receptor agonist.7-9 Respi-
   Received February 28, 1990; accepted July 19, 1990.             ratory activity also was monitored because neurons

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1268    Circulation Research Vol 67, No 5, November 1990

reached by diffusion of drugs topically applied to the         pledgets. In each experiment, control solutions of 2
intermediate area not only affect cardiovascular func-         ,ul of the solvents used to dissolve DOI, ketanserin,
tion but also affect breathing.6                               N-methyl-D-aspartic acid (NMDA), and prazosin
                                                               were examined. Pledgets soaked in the solvents used
               Materials and Methods                           to dissolve these drugs did not produce significant
General                                                        changes in cardiorespiratory activity.
  Experiments were performed on adult cats of                  Stellectomy
either sex, weighing between 1.7 and 4.3 kg. Anes-                Animals were ventilated artificially with room air.
thesia was induced and maintained with a single                Crescent-shaped incisions were made bilaterally ipto
intravenous dose of a-chloralose (75 mg/kg). Rectal            the chest wall from the manubrium to the posterior tip
temperature was monitored and maintained between               of the scapula. The various muscle layers were excised,
37.00 and 38.00C with an infrared heating lamp                 revealing the rib cage. On each side, the second rib was
connected to a thermistor. The femoral artery and              isolated from the intercostal muscles and excised. Both
vein were cannulated for the measurement of arterial           stellate ganglia were then dissected away from the
blood pressure and for the systemic administration of          surrounding fascia and removed. To determine the
drugs, respectively. Lead II of the electrocardiogram          effectiveness of bilateral stellectomy in abolishing sym-
was monitored, and heart rate was determined by                pathetic neural effects on the heart, the degree of
measurement of the RR interval. The trachea was                reflex-induced sinus tachycardia produced by sodium
cannulated and fitted with a Fleisch No. 0 pneumo-             nitroprusside infused intravenously at a rate of 200
tachograph connected to a respiratory flow trans-              ,g/mVmin for 15 seconds was observed before and
ducer (HP 4730A, Hewlett-Packard Co., Waltham,                 after extirpation of the stellate ganglia. With both
Mass.). The airflow signal obtained from the trans-            stellate ganglia intact, sodium nitroprusside infusion
ducer was integrated with a Hewlett-Packard respi-             lowered mean blood pressure -31±5 mm Hg and
ratory integrator (HP 8815A) to obtain tidal volume.           raised heart rate +39±7 beats/min. After bilateral
Respiratory rate was obtained from fast tracings of            stellate ganglionectomy, sodium nitroprusside infusion
the flow signal, and respiratory minute volume was             lowered mean arterial pressure -36+ 10 mm Hg with-
calculated as the product of tidal volume and respi-           out appreciably changing heart rate (-2±+1 beats/min).
ratory rate. All respiratory and cardiovascular pa-            Hence, the lack of reflex-induced sinus tachycardia
rameters were recorded simultaneously on a Hew-                with sodium nitroprusside indicated successful stellate
lett-Packard eight-channel recorder (HP 7758B).                ganglionectomy.
   Control measurements were taken at approxi-
mately 5-minute intervals for at least 30 minutes              Experiments of Contractile Force
before the administration of drugs in each experi-                In some animals, the effects of drugs applied to the
ment. Once a drug was administered, measurements               intermediate area on contractile force of the right
were made at the time at which peak responses                  ventricle were examined in the absence and presence
occurred and then at 5-minute intervals thereafter             of bilateral stellate ganglionectomy. These animals
until cardiorespiratory activity had returned to the           were ventilated artificially with room air. A cali-
predrug level or had stabilized at a new level.                brated strain gauge arch (Warren Research Prod-
                                                               ucts, Charleston, S.C.) was sewn onto the right
Ventral Surface Application of Drugs                           ventricle and stretched by 50% of its initial length.
   To apply drugs on the intermediate area of the              Right ventricular force was measured because
ventrolateral surface of the medulla, the following            changes in systemic pressures have relatively less
procedures were performed. After a longitudinal                effect on right than on left ventricular diastolic fiber
midline incision was made in the neck, the trachea             length.11,12 The leads of the arch were connected to a
and esophagus were transected and retracted ceph-              preamplifier (model 7P1A, Grass Instrument Co.,
alad. The prevertebral muscles were bluntly dis-               Quincy, Mass.), which was coupled to a dynograph
sected away from the base of the skull, which then             recorder (model R611, Sensormedic, Anaheim,
was removed. The dura and arachnoid membranes                  Calif.). A bipolar electrode was sutured onto the
then were removed. Cottonoid wicks were placed on              right atrial appendage. The electrode served as a
all edges of the craniotomy to prevent excessive               pacing electrode and was connected to a stimulator
pooling of cerebrospinal fluid during the experiment           (Grass Instruments). Blood pressure and lead II of
and, thereby, prevented excessive dilution and spread          the electrocardiogram were monitored on a recorder
of the drug solutions.                                         (model 220, Gould Instruments, Cleveland).
   Drugs were applied to the intermediate area of the
ventral surface of the medulla with 2-mm-diameter              Microinjection Technique
pledgets of No. 2 filter paper that were soaked with 2           The ventral surface of the medulla was exposed in
,ul of drug solutions. Additional details of the proce-        the same manner described above. Double-barreled
dure and location of drug application have been                micropipettes (i.d., 0.3 mm; FHC, Inc., Brunswick,
described previously.10 Administration of 2 ,ul of drug        Me.) were pulled with a vertical pipette-puller (model
or control solutions resulted in saturation of the             700C, David Kopf Instruments, Tujunga, Calif.), and

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Mandal et al 5-HT2 Receptors and Sympathetic Outflow        1269

the tips were cut to approximately 15 ,um i.d. The              a bright green area; in some cases, tissue damage also
pipette was then mounted on a David Kopf electrode              marked the micropipette track.
manipulator. Each barrel of the pipette was connected
to polyethylene tubing and filled using negative pres-          Drugs Used and Drug Preparation
sure. One barrel was filled with a 100 mM solution of              a-Chloralose was obtained from ICN Biomedical,
L-glUtamic acid, while the other was filled with DOI,           Cleveland, and dissolved in 30 ml heated 0.9% saline.
which also contained a 1% solution of fast green                DOI was obtained from Research Biochemicals, Inc.,
(Sigma Chemical Co., St. Louis) for subsequent histo-           Natick, Mass., and was dissolved in 0.9% saline. Ket-
logical verification of the injection site.                     anserin tartrate was obtained from Research Biochem-
   Two microinjection sites were selected in evaluat-           icals and was dissolved in distilled water. N-Methyl-D-
ing the site of action of DOI. One of these sites was           aspartic acid was obtained from Sigma Chemical Co.,
the locus where microinjections of L-glutamic acid              St. Louis, and was dissolved in 0.9% saline. Prazosin
produce an increase in blood pressure, namely, the              was obtained from Sigma and was dissolved in ethanol
nucleus reticularis rostroventrolateralis.13 In that            and dimethyl sulfoxide and diluted with distilled water.
study, a volume of 200 nl made up the microinjectate.           Phentolamine (Regitine) was obtained from CIBA
Since then, our laboratory has observed effects with            Pharmaceutical Co., Edison, N.J., and was dissolved in
volumes of microinjectate as low as 10 nl. With this            0.9% saline. Propranolol was obtained from Ayerst
reduced volume, we have been able to localize better            Laboratories, New York, and was dissolved in 0.9%
this "L-glutamic acid-sensitive site." This site also           saline. Sodium nitroprusside was obtained from Elkins-
has been described by McAllen14 as the subretrofa-              Sinn, Inc., Cherry Hill, N.J., and was reconstituted and
cial nucleus (SRFn). The coordinates used for micro-            diluted in 5% dextrose.
injection into the SRFn were 6.0-7.0 mm caudal to
the foramen cecum, 4.0 mm lateral to the midline,               Statistical Analysis
and 1.0-1.5 mm below the ventral surface. With                     Data were analyzed using the two-tailed paired t
these coordinates, a micropipette was placed on each            test, the two-tailed grouped t test, or one-way analysis
side. Then, using a series of pressure injections (30           of variance with Duncan's multiple range test. The
psi, 400-1,000 msec in duration) with a pneumatic               criterion for significance was p
1270        Circulation Research Vol 67, No 5, November 1990

TABLE 1. Effect of Bilateral Application of 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on the Intermediate Area of the
Ventral Surface of the Medulla on Cardiorespiratory Function
                                                                         Mean
                                                                         blood                                            Tidal                    Respiratory                        Respiratory
Experimental                           Experimental                     pressure              Heart rate                 volume                          rate                        minute volume
group                          n         condition                      (mm Hg)               (beats/min)                   (ml)                 (breaths/min)                             (ml/min)
Control                        8    Control (initial                    110+11                  209± 12                     42+4                     12±1                                   536+65
  (DOI 30                               values)
   ,ug/side)                        Maximal change                      +39+7*                  +8+5                     +12±5*                       -5+0*                               -163+38*

Ketanserin                     7    Baseline values                     116+7                  172+12                       27+1                       12+2                                 316+44
  pretreatment                        after ketanserin
  (25 rg/side)                      Maximal change                      +10±3*t                   0+2                      +1±1                       -1±It                                -31---22t
                                      produced by
                                      DOI (30
                                        ,g.g/side)
   Values are expressed as mean+SEM.
   *p
Mandal et al 5-HT2 Receptors and Sympathetic Outflow                 1271

 Tidal
           30
                                                                                                                     P1
Volume                 /                                             /                                              1,!!
  (ml)                                                                                                               11
                                                                                                               11              1

                               1 aec               1 whi

 Tracheal
 Air   Flow

         200_

 Blood            L                                               NNNNNNNN      K>NNNKN
1272          Circulation Research Vol 67, No 5, November 1990

TABLE 2. Effect of Ketanserin Pretreatment on the Cardiorespiratory Effects Elicited by Bilateral Application of N-Methyl-D-aspartic Acid
(NMDA) to the Intermediate Area of the Ventral Surface of the Medulla
                                                      Mean blood                           Tidal         Respiratory         Respiratory
Experimental                   Experimental            pressure          Heart rate       volume             rate           minute volume
group                n           condition             (mm Hg)          (beats/min)         (ml)        (breaths/min)          (ml/min)
Control              3     Control (initial             101+3             195+9             23+2            14+2                319+53
  (NMDA 11                   values)
  ,rg/side)                Maximal change              +45 +2*            +31±+ 6*          -1±+-2            0+1              -17+13

Ketanserin           4     Baseline values after      101+1                172+12           27+1             16+2               433+56
  pretreatment               ketanserin
  (25 pg/side)             Maximal change             +42±12*             +25+16           +12±6            +1+1              +239±130
                             produced by
                             NMDA
  Values are expressed as mean±SEM.
  *p
Mandal et al 5-HT2 Receptors and Sympathetic Outflow                         1273

TABLE 4. Effect of Various Pharmacological and Surgical Interventions on Cardiorespiratory Effects of 1-(2,5-Dimethoxy-4-iodophenyl)-
2-aminopropane (DOI) Applied to the Intermediate Area of the Ventral Surface of the Medulla
                                                                        Mean
                                                                        blood                           Tidal                      Respiratory     Respiratory
Experimental                                      Experimental         pressure         Heart rate     volume                          rate       minute volume
group                              n                condition          (mm Hg)          (beats/mia)      (ml)                     (breaths/min)      (ml/min)
Bilateral cervical                 3          Baseline values after      87±11            174±7         34±8                          11±2            317±39
  vagotomy                                      vagotomy
                                              Maximal change           +41±6*             -6±9             +1±2                      -1±1t          -40±32
                                                produced by DOI
                                                (30 pg/side)
Phentolamine                        3         Baseline values after      68±13            250±0             38±2                      19±4           690±134
  pretreatment                                  phentolamine
  (5 mg/kg)                                   Maximal change           +31±5*            -18±8             +1±2                      -8±1*         -290±64*
                                                produced by DOI
                                                (30 pg/side)
Propranolol                         5         Baseline values after     128±12            179±7             33±4                      16±2           560±131
  pretreatment                                  propranolol
  (1 mg/kg i.v.)                              Maximal change           +11±3*t           -13±7             -2±2                      -7±2           -271±112
                                                produced by DOI
                                                (30 ,g/side)
Bilateral stellate                  3         Baseline values after      76±11            156±13
  ganglionectomy                                ganglionectomy
                                              Maximal change           +18±4*t            +4±3
                                                produced by DOI
                                                (30 ,g/side)
  Values are expressed as mean±SEM.
  *p
1274    Circulation Research Vol 67, No 5, November 1990

                                                      Stellate Gaanglia Intact.

                                                *     Stollate Ganglia Removed.
       6-          p
Mandal et al 5-HT2 Receptors and Sympathetic Outflow                1275

TABLE 5. Effect of Ketanserin Pretreatment (i.e., Ketanserin Applied to the Intermediate Area) and Propranolol Pretreatment (i.e.,
Propranolol Administered Intravenously) on Cardiorespiratory Effects of 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) Admin-
istered Intravenously
                                                          Mean blood                       Tidal       Respiratory        Respiratory
                                     Experimental          pressure        Heart rate     volume           rate          minute volume
Experimental group        n            condition           (mm Hg)         (beats/min)      (ml)      (breaths/min)         (ml/min)
DOI (100 ,g/kg i.v.)      3      Control (initial           142±4               210±+16    36±+10         12±1               449± 124
                                   values)
                                 Maximal change             +33±2*              -8+12     -11±10         +1±1                 -116±146

Animals pretreated        2      Control (values after       64±4               159±20     31±0           13±0                 382±7
  with ketanserin                  ketanserin)
  (25 pg/side) and
  givenDOI (100                  Maximalchange              +47+2               -9±2      -7±2           +6±1                 +80±10
  ,ug/kg i.v.)

Animals pretreated        3     Control (values after       98±30               127±13     31±8           13±5                 312±58
  with propranolol                propranolol)
  (1 mg/kg i.v.) and
  given DOI (100                Maximal change            +38±8*                -12±10    -8±0*               0±1             -80±54
  pg/kg i.v.)
  Values are expressed as mean±SEM.
  *p
1276     Circulation Research Vol 67, No 5, November 1990

                   LL         200i

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                                                                                            1 sec.
                                0                       1 min.

                                       HR: 194                                              HR: 200

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                                        20 ni OF L-GLUTAMIC ACID                  20 nl OF L-GLUTAMIC ACID
                                               ON LEFT SIDE                              ON RIGHT SIDE

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                                                                 1 ug OF DOI ON LEFT SIDE    1 ug OF DOI ON RIGHT SIDE
FIGURE 5. Representative experiment showing the use of microinjection ofL-glutamic acid to locate the subretrofacial nucleus on
each side (top panel) and the effects of bilateral microinjection of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 1
 pg/side) into the subretrofacial nucleus on blood pressure and heart rate (HR, bottom panel).

would counteract the pressor effect of intravenously                                  sequence   of microinjections is shown in Figure 5. As
administered DOI. Data obtained from three animals                                    can  be seen, microinjection of L-glutamic acid on
are shown in Table 5 and indicate that propranolol                                    each side evoked an immediate increase in blood
pretreatnent does not counteract the pressor effect                                   pressure, which was brief in duration. When arterial
produced by a bolus intravenous injection of DOI.                                     blood pressure had stabilized, DOI was microin-
                                                                                      jected bilaterally into the same site in a dose of 1
Microinjections of DOI Into the                                                       gg/site. Immediately after microinjection of DOI,
Subretrofacial Nucleus                                                                there was a striking increase in arterial pressure:
   In an attempt to better localize the site of the                                   mean blood pressure increased from 78 to 112
pressor effect of DOI within the ventrolateral me-                                    mm Hg, producing an increase of 34 mm Hg. The
dulla, this agent was microinjected into a major                                      location of the micropipette tips in this experiment is
nucleus that influences central sympathetic outflow                                   illustrated in Figure 6. As can be seen, the injection
and that can be reached by diffusion of drug initially                                sites were within the SRFn.
placed on the intermediate area of the ventrolateral                                     In subsequent experiments, lower doses of DOI
medulla. This nucleus is the SRFn.14 Coordinates                                      were microinjected into the SRFn of cats that had
have been established for locating this nucleus (see                                  undergone cervical vagotomy. The doses were 100
"Materials and Methods"). Along with these coordi-                                    and 300 ng/site, and the data obtained are shown in
nates, an additional guide for locating the SRFn is to                                Table 6. These two doses of DOI appeared to
find the site where L-glutamic acid microinjection                                    produce a dose-related increase in mean arterial
produces a rapid and marked increase in arterial                                      pressure. The response observed with the 300 ng/site
blood pressure. Hence, these experiments were con-                                    microinjection was similar in magnitude to that ob-
ducted first by microinjecting 20-60 nl of a 100 mM                                   served after topical application of 30 jig/side DOI to
solution of L-glutamic acid as a test agent for locating                              the intermediate area (compare data in Table 6 with
the SRFn on each side. After the blood pressure and                                   data in Table 1). The increases in mean arterial
heart rate had returned to baseline values, the effects                               pressure produced by microinjection of 100 and 300
of bilateral microinjections of DOI were then deter-                                  ng/site were not associated with significant changes
mined. A representative experiment illustrating this                                  in heart rate (Table 6).

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.t it.t
1278     Circulation Research Vol 67, No 5, November 1990

applied topically to the intermediate area of the ventral        with the intermediate area. The major nucleus asso-
surface of the medulla. These data also are summarized           ciated with the intermediate area and shown to
in Table 6 and indicate that topical application of 100,         influence sympathetic outflow in the cat is the
300, and 1,000 ng/side (i.e., 1 ,ug/side) had no signifi-        SRFn.14 Hence, experiments were performed to de-
cant effect on mean arterial blood pressure.                     termine whether the pressor effect of topically ap-
                                                                 plied DOI was due to diffusion to this nucleus. This
                       Discussion                                was tested by microinjection of DOI bilaterally into
   The major new findings from our study are as                  the SRFn in doses that were inactive after topical
follows: 1) stimulation of 5-HT2 receptors in the                application to the intermediate area and then observ-
ventrolateral medulla, presumably in the SRFn, in-               ing the same spectrum of cardiovascular effects that
creases sympathetic outflow, resulting in an increase            were obtained after topical application of DOI to the
in arterial blood pressure; and 2) activation of 5 -HT2          intermediate area in a dose of 30 ,ug/side. In contrast,
receptors in the ventrolateral medulla results in a              bilateral microinjections of 300 ng/site DOI into the
nonuniform increase in sympathetic outflow. The                  parapyramidal region had no effect on blood pres-
outflow appears to be increased primarily to ventric-            sure. As mentioned earlier, the parapyramidal region
ular muscle with either no or relatively minor                   also has been shown to influence sympathetic out-
changes in outflow to the sinus node and vascular                flow.17-19 Our data strongly indicate that the site of
beds.                                                            action of DOI to increase sympathetic outflow and
   Evidence that activation of 5-HT2 receptors in the            raise arterial blood pressure is the subretrofacial
ventrolateral medulla increases sympathetic outflow,             nucleus, not the parapyramidal region.
resulting in an increase in arterial blood pressure, was            Evidence that activation of 5-HT2 receptors in the
obtained initially in our previous studies with 5 -HT            ventrolateral medulla results in a nonuniform in-
applied to the intermediate area of the ventral sur-             crease in sympathetic outflow is based on several
face of the medulla.1 In that study, topical applica-            findings, and they are as follows. 1) Both topical
tion of 5-HT to the intermediate area produced a                 application of DOI to the intermediate area and
small but statistically significant hypotensive effect.          microinjection of DOI into the subretrofacial nucleus
By blocking 5 -HT2 receptors at this site, hypotension           in vagotomized animals cause an increase in arterial
resulting from topical application of 5-HT was en-               blood pressure without any significant change in
hanced significantly. These data implied that 5-HT               heart rate, indicating that DOI increases sympathetic
was activating 5-HT2 receptors in the intermediate               outflow but not to the sinus node. 2) The pressor
area, resulting in an increase in arterial blood pres-           effect of DOI applied to the intermediate area is
sure. With the introduction of a specifically acting             counteracted either by systemic propranolol pre-
agonist for the 5 -HT2 receptor, namely DOI, we were             treatment or by bilateral stellate ganglionectomy but
able to test directly whether activation of this recep-          not by systemic phentolamine pretreatment. These
tor subtype would result in an increase in arterial              observations indicate that DOI increases sympathetic
blood pressure. Indeed, topical application of DOI to            outflow to the heart (with the exception of the
the intermediate area did produce a consistent rise in           sinoatrial node) but not to the vasculature. 3) The
arterial blood pressure. Similar results of DOI in               pressor effect of topically applied DOI is associated
anesthetized cats recently have been reported by                 with an increase in cardiac contractile force, and
King and Holtman.21                                              bilateral stellate ganglionectomy counteracts both
   Evidence that the effect of DOI was mediated                  DOI-induced increases in cardiac contractile force
through activation of 5 -HT2 receptors was obtained              and in arterial blood pressure. This latter result
using the 5-HT2 receptor antagonist drug ketanserin.28           provides confirmatory evidence that DOI is selec-
Topical application of ketanserin to the intermediate            tively increasing sympathetic outflow to the heart
area prevented the pressor effects of subsequent ad-             (with the exception of the sinoatrial node). 4) In
ministration of DOI to the same site. The selectivity of         contrast to the pressor effect of DOI, the pressor
ketanserin for blocking the 5-HT2 receptor was dem-              effect of NMDA topically applied to the intermediate
onstrated by showing that a dose of ketanserin that              area is associated with both an increase in heart rate
blocked the pressor effect of DOI did not block the              and an increase in cardiac contractile force that is not
pressor effect of NMDA applied topically to the inter-           counteracted by bilateral stellate ganglionectomy.
mediate area. Because ketanserin also blocks a1-                 This latter finding in addition to the earlier findings
adrenoceptors,24-26,28 it was important to show that the         of McAllen29 indicates that activation of neurons in
interaction between ketanserin and DOI was not due               the ventrolateral medulla (intermediate area) causes
to the a1-adrenoceptor blocking property of ketanserin.          widespread increases in sympathetic outflow,
To this end, it was noted that the prototypic a1-                whereas activation of 5-HT2 at the same site causes a
adrenoceptor antagonist, prazosin, did not antagonize            selective increase in sympathetic outflow to the heart
the DOI-induced pressor response.                                (i.e., to the ventricles). Based on these findings, we
   The lack of a pressor effect of DOI topically                 postulate that one reason neurons in the central
applied to either the rostral area or the caudal area            nervous system can be selectively engaged to perform
of the ventral surface of the medulla indicated that             a physiological function is because of the pattern of
DOI was exerting its effect at a nucleus associated              distribution of neurotransmitter receptors on neu-

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Mandal et al 5-HT2 Receptors and Sympathetic Outtlow                 1279

rons, such as the 5-HT2 receptor on certain neurons             that this central hypotensive effect is due to the
within the SRFn.                                                actions of ketanserin as an a,-adrenoceptor antago-
   Other investigators have obtained data indicating            nist rather than as a 5-HT2 receptor antagonist.36'37
that systemic administration of DOI can increase                In addition, ketanserin has been shown to have
central sympathetic outflow3,4'30 and arterial blood            relatively high affinity for receptors other than the
pressure.3,4,29-32 Heart rate was increased slightly in         5-HT2 receptor and the a1-adrenoceptor,2' and these
the initial study by McCall and colleagues,3 but not in         other receptor effects might contribute to its central
the later study4 in which a similar dose range of DOI           hypotensive action. Further studies with newer and
was used. Other investigators have reported that                more selective 5 -HT2 antagonists, such as LY-
DOI, in doses that exert a pressor effect, does not             53857,25,39 and their effects at the ventrolateral me-
increase the heart rate.30-32 Most important, McCall            dulla need to be performed to resolve this important
and colleagues3 reported that DOI increases sympa-              question.
thetic outflow through a central nervous system site               In our study, we also observed changes in respira-
of action. Their evidence was based on the finding              tory activity after topical application of DOI to the
that DOI given intravenously increases sympathetic
discharge in preganglionic splanchnic fibers in                 intermediate area. Specifically, these changes were an
baroreceptor-denervated cats. In our study, we ob-              increase in tidal volume and a decrease in respiratory
served that intravenously administered DOI in-                  rate. This decrease in respiratory rate has been de-
creased the arterial pressure. This finding raises the          scribed in the study by King and Holtman.21 Previ-
question as to whether intravenously administered               ously, we reported that topical application of drugs
drug acts at a site in the ventrolateral medulla to             that activate 5--HT1A receptors to the intermediate
exert a pressor effect. To test this point, we repeated         area decreases tidal volume and increases respiratory
our intravenous studies under conditions for which              rate.1'40A4l Therefore, just as activation of 5-HT2 recep-
the pressor effect of topically applied DOI to the              tors and activation of 5-HT1A receptors in the inter-
ventrolateral medulla was inhibited. These condi-               mediate area have opposing effects on blood pressure,
tions were after topical application of ketanserin to           activation of these two subtypes of serotonergic recep-
the intermediate area of the ventrolateral medulla              tors also has opposing actions on respiratory activity.
and after intravenous administration of propranolol.            Furthermore, the respiratory effects of topically ap-
Under both conditions, intravenously administered               plied DOI were lost after microinjection of DOI in the
DOI produced a striking increase in arterial blood              SRFn. Therefore, it appears that the central site of
pressure. Based on these findings, we conclude that             action for the respiratory effects of DOI is not the
intravenous DOI is not exerting a pressor effect by             SRFn but some other area. More studies need to be
stimulating 5 -HT2 receptors in the region of the               performed to elucidate the site of DOI's respiratory
ventrolateral medulla.                                          effects.
   According to Dabire and colleagues,31 DOI pro-                  In summary, our data indicate that there is a central
duces a greater increase in arterial blood pressure in          pathway for selective sympathoexcitation of the heart,
the pithed rat as compared with the intact rat. This            leading to an increase in cardiac inotropy without an
result indicates that the central nervous system is not         increase in cardiac chronotropy. The pathway for this
required for the action of intravenous DOI to raise             selective sympathetic outflow to the cardiac ventricles is
arterial blood pressure. Indeed, it has been shown that         mediated by activation of 5-HT2 receptors on neurons
there are 5-HT2 receptors on vascular tissue and that           located within the ventrolateral medulla, most likely
activation of these receptors results in vasoconstric-          within the SRFn. Although it has been shown that it is
tion.33-35 The question we have not addressed in our            possible to selectively increase sympathetic outflow
study is whether the DOI-induced increase in central            from the SRFn to different vascular beds,'4 to our
sympathetic outflow after systemic administration is            knowledge, our report is the first that documents
due to an interaction of DOI with 5-HT2 receptors               selective sympathoexcitation to different areas within a
located in the ventrolateral medulla. We suspect that           specific organ, namely, the heart.
this is the case; however, experiments need to be                                        References
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      665-674                                                           heart rate * serotonin receptors * subretrofacial nucleus

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Stimulation of serotonin2 receptors in the ventrolateral medulla of the cat results in
                      nonuniform increases in sympathetic outflow.
                    A K Mandal, K J Kellar, W P Norman and R A Gillis

                                       Circ Res. 1990;67:1267-1280
                                      doi: 10.1161/01.RES.67.5.1267
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