The pressor response to spinal cholinergic stimulation in spontaneously hypertensive rats

Brain Research Bulletin,

0361.9230&O$3.00 + .OO

Vol. 25, PP. 69-74. c PergamonPress pk. 1990.Printedin the U.S.A

The Pressor Response to Spinal Cholinergic Stimulation in Spontaneously Hypertensive Rats JERRY J. BUCCAFUSCO’ AND VENERA MAGRI’ Department of Pharmacology and Toxicology Department of Psychiatry and Health Behavior Oedipal College of Georgia, Augusta, GA 3091~-~300 and The Veterans Administration Medical Center, Augusta, GA 30910

Received 18 December 1989 BUCCAFUSCO, J. J. AND V. MAGRI’. The pressor response to spinaf cholinergic stimulation in spontaneously hypertensive ruts. BRAIN RES BULL 25(l) 69-74, 1990.-Several laboratories have demonstrated that central cholinergic stimulation in spontaneously hypertensive rats (SHR) results in an exaggerated pressor response as compared to normotensive (NT) controls. Recent studies in this laboratory have demonstrated a spinal cholinergic pressor system in the NT rat. The purpose of this study was to determine whether the pressor response to spinal cholinergic stimulation is enhanced in SHR. In freely moving rats, intrathecal injection of neostigmine or carbachol (1-5 kg) produced a dose-related hypertensive response in both strains of rats. While both agonists produced similar maximal increases in blood pressure in NT rats, the pressor responses to both agonists were significantly greater in SHR. The tachycardic responses to IT injection of cholinergic agonists were also signi~cantiy greater in SHR. These differences were more apparent at the lower doses where, for example, the pressor response to 1 p.g of agonist in the SHR was increased by 123% and 109% of the response in NT rats for carbachol and neostigmine, respectively. Since both direct and indirect acting agonists produced greater responses in SHR, and spinal depletion of acetylcholine did not reduce blood pressure in SHR, it is most likely that spinal cholinergic systems ascend to higher centers to elicit pressor responses. In the case of the SHR, these higher centers may be supersensitive to cholinergic stimulation. Spontaneously hypertensive rat

Spinal cord

Carbachol

Neostigmine

ACTIVATION of central cholinergic neurons evokes a pressor response in several animal species, as well as man [(2,8) for review]. This pressor response can be produced by central muscarinic receptor stimulation or through inhibition of brain cholinesterase. Since the in~oduction of the s~nt~eously hypertensive rat (SHR) strain in the early 1970’s it has been recognized that certain central cholinergic systems may be abnormal, and in fact, participate in the development and mainten~ce of hy~~ension in this strain (25). SHR and other rat models of hypertension are known to be particularly sensitive to central cholinergic stimulation (3, 13, 14, 1.5, 18). Such sensitivity is not due to heightened vascular reactivity (3). Sensitivity to central cholinergic stimulation is easily demonstrated following intravenous (IV) injection of the centrally acting cholinesterase inhibitor, physostigmine which evokes an enhanced hy~~nsive response in SHR (3, 14, 18). Recent studies in this laboratory have demonstrated that this exaggerated pressor response to physostigmine is not mirrored folIowing IV injection of the direct acting agonist, arecoline ( 18). While arecoline is capable of stimulating central muscarinic receptors, it does not require (as does physostigmine) the partici-

Blood pressure

pation of endogenous ace~lchol~e release to evoke its hypertensive response (18). We concluded, therefore, that the exaggerated hypertensive response observed in SHR following IV injection of physostigmine is due to enhanced release of brain acetylcholine, rather than muscarinic receptor supersensitivity. This hypothesis is strengthened through our recent finding of enhanced neuronal cholinergic activity in the medulla of SHR as measured biochemically (22). Thus direct and indirect acting cholinergic agonists may be employed to localize centers of enhanced cholinergic activity in the SHR; and, to determine whether the exaggerated activity is due to supersensitivity of muscarinic receptors, or to enhanced release of the neurotransmitter. In addition to the exaggerated pressor response to cholinesterase inhibitors observed for hypertensive animals, the reverse is also true. That is, in conscious, freely moving rats, interference with central cholinergic function results in an antihypertensive response in hypertensive, but not in normotensive animals. This has been demonstrated in SHR (1, 3, 1 l), DOCA-salt hypertension, Grollman hypertension, aortic coarctation-induced hypertension (11) and the Dahl salt-sensitive hypertensive strain (15).

‘Requests for reprints should be addressed to Dr. Jerry J. Buccafusco, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300.

69

70

BUCCAFUSCO

Studies from this laboratory have demonstrated that a hypertensive response is observed following intrathecal injection of cholinergic agonists in unanesthetized, freely moving normotensive rats (9, 16, 19). Although, this response is mediated by spinal muscarinic receptors, depletion of spinal levels of acetylcholine do not affect the pressor response to intrathecal carbachol. Alternately, depletion of acetylcholine within the medulla does block the pressor response to intrathecal injection of carbachol(9). Also, high spinal transection eliminates the pressor response to both carbachol and neostigmine (19). Based upon all of our current findings, it appears that stimulation of spinal muscarinic receptors results in activation of an ascending system interacting with a second medullary cholinergic pathway which subsequently leads to the development of the hypertensive response. The purpose of this study was to examine the hypertensive responses to intrathecal injection of the indirect acting agonist, neostigmine, and the direct acting agonist, carbachol, in SHR and NT rats; and to determine whether depletion of spinal acetylcholine in SHR results in an antihypertensive response.

AND MAGRI‘

NORMOTENSNE

20

90 min

after

4.0

60

so

70

so

90

i.t. Neostigmine

FIG. 1. The change in mean arterial pressure (MAP) following intrathecal injection of neostigmine in freely moving normotensive rats. Each value represents the mean 2 S.E.M. of 5-7 experiments.

METHOD

This study was performed using conscious, freely moving rats in their home cage environment. Male, spontaneously hypertensive rats (SHR) and aged-matched normotensive out-bred Wistar (NT) rats were obtained from Harlan Sprague-Dawley, Indianapolis, IN and housed in an environmentally controlled room having a 12-hr light-12-hr dark cycle. Standard rat chow (Wayne Rodent Blox) and tap water were supplied on an unlimited basis. The animals were 16-20 weeks of age at the time of the experiment. Although the genetically related strain Wistar Kyoto (WKY) rat is generally employed as the normotensive control for the SHR, we have demonstrated previously that there is no significant difference in the baseline levels of either blood pressure or heart rate, or in the profile or magnitude of the pressor response to central cholinergic stimulation between out-bred Wistar and WKY rats (18). In addition, central cholinergic neuronal activity was previously demonstrated to be similar between both strains of rats (22). Therefore, for economical reasons, out-bred Wistar controls were employed in this study. Indwelling Intrathecal (IT) Catheters Rats were anesthetized with 65 mg/kg, IP, of sodium methohex&al and placed in a stereotaxic frame. Catheterization of the spinal subarachnoid space was performed as described previously (24) by inserting a sterile, normal saline-filled polyethylene (PE 10) catheter 5 5 cm caudal to a midline nick in the atlantooccipital membrane and terminating in the T7-T8 level of the spinal cord. The rostra1 end of the catheter was plugged with a 30-gauge stainless steel wire and anchored to the skull with acrylic cement. Animals were allowed 5 days to recover prior to any additional procedures. Only normally moving, healthy animals were employed in subsequent experiments. Intrathecal (IT) injections (5 ~1) were delivered via a 50+,1 syringe over 15 set using a constant speed syringe pump. The injection was followed with a lo-pl saline flush to clear the catheter of drug. Upon completion of the experiments, catheter placement was confiid by dye injection and dorsal laminectomy. Furthermore, our earlier studies have demonstrated that tissue cholinesterase inhibition following IT injection of neostigmine is restricted to spinal regions (16). Preparation for Arterial Blood Pressure Recordings Rats were anesthetized with methohexital and a midline abdominal incision made to expose the left iliac artery. A polyeth-

ylene (PE 50) catheter, filled with heparinized (20 units/ml) saline was inserted so that the tip of the catheter terminated in the base of the abdominal aorta, below the origin of the renal arteries. The opposite end of the catheter was plugged with 22-gauge wire and directed subcutaneously to emerge at the back of the neck. Following surgery the animals were returned to their home, clear plastic cages (45 X 25 x 20 cm), and the catheter passed through a

spring support and connected to a water-tight swivel cannula mounted 30 cm above the cage floor. This method allowed the chronically catheterized rat unrestricted movement to all areas of the cage while a constant infusion (8 ml/day) of heparinized saline (20 units/ml) maintained the patency of the catheter. Animals were allowed to recover for 48 hr prior to intrathecal injections. At the time of the experiment, arterial catheters were connected to pressure transducers coupled to a Western Graphtec, Mark 7 polygraph recorder for continuous arterial pressure recording. Heart rate (HR) was determined by a cardiotachometer triggered from the pressure pulses. The cardiovascular responses to cholinergic agonists are expressed as change from preinjection levels obtained when animals were resting quietly. Mean arterial pressure (MAP) was calculated as (pulse pressure/3) + diastolic pressure. Statistics Comparison between the means of several populations was performed using a one- or two-way ANOVA or an ANOVA for

repeated measures, and the differences considered significant at the p
Blood Pressure Changes to IT Injection of Cholinergic Agonists

Resting levels of MAP in 40 SHR and 40 NT rats averaged 1512 5 and 105 r 3 mmHg, respectively. The means from the two groups were significantly different pCO.01. Intrathecal injection of neostigmine (1, 5 and 10 p,g) produced a dose-dependent hypertensive response in NT rats (Pig. 1). The pressor response developed slowly, especially at the lowest dose and became maximal between 10 and 15 min after injection. At this time, MAP increased to about 40 mmHg after the highest dose. In SHR, the

SPINAL CHOLINERGIC

HYPERTENSION

71

IN SHR

NOFiWTENsIvE 00

00

40

SO

20

10

0 0

10

90

a0

min after

40

50

00

70

I RI

a0

response

to neostigmine

10

20

was

also

dose

30

dependent,

but

blood pressure increased more quickly, reaching maximal levels prior to 10 min after injection (Fig. 2). Also, the magnitude of the pressor response was significantly greater in SHR. This was particularly evident at the lower doses. Note that for the 1 kg dose, in NT rats the pressor response had returned to control level within 1 hr after injection. In SHR, however, the pressor response was still significantly elevated at 90 min. Data for the magnitude of the maximal increase in MAP (determined for each animal) is presented in Table 1. As indicated, the pressor response obtained following IT injection of neostigmine in SHR was up to 109% greater compared with NT controls. The differences between strains became smaller as the dose increased, however, this most likely reflects a ceiling effect. Intrathecal injection of carbachol (1, 5 and 10 kg) produced a dose-dependent hypertensive response in NT rats (Fig. 3). The

40

after

min

i.t. Neostigmine

FIG. 2. The change in mean arterial pressure (MAP) following intrathecal injection of neostigmine in hypertensive (SHR) rats. Statistical comparisons with the data derived from NT controls (Fig. 3) are as follows, 1 p,g neostigmine: Between Groups, F(l,l l)= 10.91, ~~0.01; 5 kg neostigmine: Between Groups, F(1.15) = 7.63, ~K0.05, Interaction, F(l4.210) = 1.78, pcO.05; 10 p,g neostigmine: Between Groups, F(1,12)=5.16, rKO.05, Interaction, F(14,168)=2.22, pcO.01.

pressor

0

SO

Bo

70

80

w

i.t. Carbachol

FIG. 3. The change in mean arterial pressure (MAP) following intrathecal injection of carbachol in freely moving normotensive rats. Each value represents the mean 2S.E.M. of 5-7 experiments.

pressor response developed much faster than neostigmine reaching maximum between 1 and 5 min after injection. (This difference in onset between carbachol and neostigmine presumably represents the direct and indirect nature of the agonist activities for the two compounds.) At the peak of the response, MAP increased to about 40 mmHg after the highest dose. In SHR, the pressor response to carbachol was also dose dependent (Fig. 4), and like neostigrnine, the magnitude of the pressor response was significantly greater than that observed for the NT strain. Again, this was particularly evident at the lower doses (Table 1). Heart Rate Changes to IT Injection of Cholinergic Agonists Resting levels of heart rate (HR) in 40 SHR and 40 NT rats averaged 352 2 9 and 364 ? 7 beats/min, respectively. The means from the two groups were not significantly different. IT injection of carbachol was associated with a significant increase in HR in NT rats which on the average did not exceed 25% of resting values

HYPERTENSIVE so T

TABLE 1 MAXIMAL CHANGE IN BLOOD PRESSURE FOLLOWING INTRATHECAL INJECTION OF CARBACHOL OR NEOSTIGMINE

Dose

1 kg

5 kg

10 pg

19.4 t 3.67 43.3 k 7.03 123%*

28.2 2 5.95 41.8 2 2.90 48%

41.0 2 2.70 52.5 k 6.10 28%

l/&g o-o 5LLg O-0 lO/lg A-A

5”

Carbachol NT SHR % Difference Neostigmine NT SHR % Difference

0

16.6 + 3.70 34.7 i- 5.70 109%*

38.7 k 3.70 57.9 2 3.70 50%*

43.4 2 6.50 60.8 + 7.50 40%

Each value represents the maximal change (observed for each rat) in mean arterial pressure (mmHg) from preinjection resting levels. % Change indicates the percentage increase for SHR compared with NT values. Data are presented as the mean 2 S.E.M. of 5-7 experiments. *Significantly different from respective NT mean, p
0

10

20

I 90

min

40

after

SO

it.

00

70

80

00

Carbachol

FIG. 4. The change in mean arterial pressure (MAP) following intrathecal injection of carbachol in hypertensive (SHR) rats. Statistical comparisons with the data derived from NT controls (Fig. 1) are as follows, 1 p,g carbachol:BetweenGroups,F(l,l2)= 17.l,p
72

BUCCAFUSCO

TABLE 2

HYPERTENSIVE

MAXIMALCHANGEINHEARTRATEFOLLOWINGINTRATHECAL INJECTIONOFCARBACHOLORNEOSTIGMINE

2 $

Dose

1

AND MAGRI‘

5

IJG

20

T

16

1

10 6

10 lJ4

I%

4

Carbachol NT SHR % Difference

90.0 -c 20.8 143.6 r 9.2 60%*

63.3 2 15.0 111.7 Y!Z16.4 76%*

80.0 + 13.0 160.0 + 9.3 loo%*

Neostigmine NT

43.1 k 16.9

58.3 k 17.1

41.7 Yz 13.3

SHR % Difference

138.3 f 2.5* 220%*

80.0 Z!Y14.5 37%

73.3 2 15.2 76%

1

0

4

o_o’

0

yj

_ _ --Q--o----,_Q,

p-+---+-6

A

-m_l

g PJ z e g

Each value represents the maximal change (observed for each rat) in

heart rate (beats/rnin)from preinjectionresting levels. % Change indicates the percentage increase for SHR compared with NT values. Data are presented as the mean2S.E.M. of 5-7 experiments. *Significantly different from respective NT mean, pcO.05.

E 3 3u

40~

0

10

20

a0

40

a0

00

70

a0

00

mh after HC-3.20~. Lt.

(Table 2). Also, these increases did not appear to be dose related. Nevertheless, in SHR, the tachycardic response was significantly exaggerated compared with normotensive values at each dose. IT injection of neostigmine was also associated with an increase in HR, however, the responses were not as great as with carbachol (Table 2). Consistent with the response to carbachol, no doseresponse relationship was obtained, although values observed for SHR were greater (comparisons for the two higher doses did not reach the level of significance) than those for NT rats. For both neostigmine and carbachol, behavioral symptoms accompanied the cardiovascular changes. These included tremor, scratching, tail biting, chewing, grooming motions and head bobbing. These did not occur in all animals or at all times and there was no attempt at quantitation. These symptoms were, in general, more prevalent following neostigmine, however, both hypertensive and normotensive animals appeared to respond similarly in this regard. Blood pressure changes were independent of behavioral activity, although, HR was somewhat more labile. This might explain the lack of a dose-response relationship and the marked variability for the HR changes following injection of cholinergic agonists. Effect of Spinal Acetylcholine

Depletion With HC-3

At least two possibilities might account for the observation that the pressor response to IT injection of carbachol was also enhanced in SHR. First, that spinal muscarinic receptors were supersensitive and that the heightened response was due to a postsynaptic alteration. Alternately, it was possible that the exaggerated response was not being mediated at the site of the injection. To distinguish between these two possibilities, 20 pg of hemicholinium-3 [a dose demonstrated to produce maximal depletion of brain acetylcholine (3). and inhibition of the pressor response to IT injection of neostigmine (16)] was administered intrathecally in SHR rats. If the exaggerated pressor responses to the cholinergic agonists were being mediated locally, then interference with local cholinergic transmission [as has been demonstrated for higher centers ( 1,3,1 l)] shouldresult in an antihypertensive response. As indicated in Fig. 5, however, HC-3 failed to significantly alter resting levels of MAP and HR in SHR. DISCUSSION

In normotensive rats the pressor response to IT injection of

FIG.5.The change in mean arterial pressure (MAP) and heart rate (HR) following intrathecal injection of 20 kg of hemicholinium-3 (HC-3) in freely moving spontaneously hypertensive rats. There was no significant change in MAP or HR over the observation period. Each point represents the mean 2 S.E.M. derived from 6 animals.

neostigmine is mediated at the level of the thoracic spinal cord, through inhibition of local cholinesterase and through stimulation of muscarinic receptors. The response is also mediated through local release of acetylcholine since HC-3 pretreatment significantly reduces the pressor response. In contrast, the pressor response to IT injection of carbachol is mediated through direct stimulation of local spinal muscarinic receptors, independent of cholinergic presynaptic mechanisms (16). Despite the patticipation of spinal cholinergic neurons in these responses, transection of the spinal cord eliminates the pressor response to IT injection of both agonists (19). The lack of effect of spinal muscarinic receptor stimulation by carbachol in the transected rat suggested that the pressor response to spinal stimulation was mediated through an ascending pathway rather than through direct activation of spinal sympathetic centers (9, 16, 19). Our most recent experiments have also demonstrated that this ascending pathway interacts with a second medullary cholinergic pathway since, 1) intracistemal (but not IT) injection of HC-3 blocked the pressor response to IT injection of carbachol (9); and 2) intracistemal injection of clonidine [which blocks the release of endogenous acetylcholine (4-7, 17) and thus the pressor response to indirect, but not direct cholinergic agonists (9)] also blocked the pressor response to IT injection of carbachol. The present results demonstrate that, as with higher brain centers, inhibition of spinal cholinesterase evokes an exaggerated hypertensive response in SHR compared with NT controls. This response was greater in magnitude, earlier in onset and longer in duration in the hypertensive strain. It was somewhat surprising, however, to observe an exaggerated pressor response following IT injection of carbachol in SHR in view of our previous findings that direct acting agonists are usually not effective in this regard following other routes of administration (18). In a recent pharmacological analysis of the enhanced pressor response to cholinergic agonists in SHR, we demonstrated that activation of pressor areas in the hypothalamus and medulla with cholinergic agonists produced hypertensive responses in SHR and NT rats. However, activation of only the medullary cholinergic system was associated

SPINAL CHOLINERGIC

HYPERTENSION

13

IN SHR

with enhanced pressor responsiveness in SHR (unpublished observation). These results are supported by neurochemical data which demonstrated greater cholinergic neuronal activity as reflected by increased sodium-dependent, high affinity choline uptake in the medulla of SHR compared with NT rats (22). Such strain differences in choline uptake were not as apparent for the hypothalmus. Therefore, it is possible that in SHR as in NT rats (9, 16, 19), stimulation of spinal muscarinic receptors activates an ascending pressor pathway which interacts with a second cholinergic medullary pressor pathway which in turn activates spinal sympathetic centers. In support of this possibility is the finding that HC-3 did not produce an antihypertensive response following IT injection in SHR whereas injections via the lateral cerebral ventricle is an effective antihypertensive treatment in several models of hypertension, including the SHR (1, 3, 11). It is not clear why the tachycardic response to IT injection of cholinergic agonists is not dose related as are the blood pressure responses. In unanesthetized rats, however, behavioral changes have a greater influence on HR than on blood pressure. We have previously described the behavioral changes accompanying the cardiovascular effects of cholinergic agonists (16,19). Also, the effects of baroreceptor function would be expected to exert full control in unanesthetized animals. Nevertheless, it is interesting that hypertensive animals exhibited a greater tachycardic response to IT injection of cholinergic agonists, and in that sense, reflected the blood pressure changes. Unlike the blood pressure, however, resting HR was not different between the two strains. Thus, these data suggest that central cholinergic control of cardiac function may also be altered in SHR. In any case, it is unlikely that the cardiac changes could account significantly for the observed differences in MAP between SHR and NT rats.

In addition to the observation that intracistemal injection of HC-3 inhibits the pressor response to IT injection of carbachol (see above) is our finding that high cervical (19), but not midcollicular transections (unpublished observation) inhibit the pressor response to IT injection of cholinergic agonists. While it is not clear how the spinal ascending cholinergic system interacts with the medullaq cholinergic system, it is possible that this occurs at the level of the rostra1 ventrolateral medulla (RVL). This region provides direct tonic vasoconstrictor activity to spinal preganglionic neurons and is defined by the presence of cells containing phenylethanolamineN-methyltransferase, the so-called C 1 region [see (2 1)] . The RVL recently has been demonstrated to receive innervation from cholinergic fibers, and stimulation of muscarinic receptors in this region in anesthetized rats evokes a hypertensive response; alternatively, muscarinic blockade results in a fall in blood pressure, even in normotensive animals (10, 12, 20, 23). Although purely speculative, it is possible that ascending spinal cholinergic afferents provide part of this innervation. In summary, the results of the present study are consistent with the presence of a powerful spinal cholinergic pressor system, one of at least 3 known central cholinergic sympathoexcitatory systems. While activation of this spinal cholinergic system results in an exaggerated pressor response in hypertensive animals, altered cholinergic activity in the SHR is most likely localized to medullary centers which receive input from the ascending spinal pathway. ACKNOWLEDGEMENTS The author would like to acknowledge

the skillful technical assistance of Ms. Laura C. Shuster. The work was supported by NM grant I-IL30046 and the Veterans Administration Research Service.

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L.; Rudy, T. A. Chronic catheterization of the spinal space. Physiol. Behav. 17:1031-1036; 1976. Neurogenic mechanisms of spontaneous hypertension. In: Femandes, M.; Kim, K. E., eds. Regulation of blood the central nervous system. New York: Grune & Stratton;