Influence of histaminergic receptors on denervated canine gracilis muscle vascular tone during endotoxemia ROBERT F. BOND, HARRY D. VILDIBILL, JR., LOUISE H. KRECH, AND JAMES C. HERSHEY Department of Physiology, University of South Carolina School of Medicine, Columbia, South Carolina 29208

BOND, ROBERTF., HARRY D. VILDIBILL, JR., LOUISE H. KRECH, AND JAMES C. HERSHEY.Influence of histaminergic receptors on denervated canine gracilis muscle vascular tone during endotoxemia. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H882-H891, 1991.-The purpose of this study was to determine if endogenously released histamine and its nonneural interaction with the HI- and Hz-histaminergic receptors in the peripheral vasculature can account for the decompensatory loss of peripheral vascular tone associated with the hypotension occurring during endotoxemia. A denervated in situ constant flow double canine gracilis muscle preparation that permitted one muscle to serve as a control (GM,) for the contralateral experimental muscle (GM,) was used. Endotoxemia was induced by intravenous infusion of 2 mg= kg-l l 30 min-l endotoxin. The specific HI and Hz antagonists diphenhydramine and cimetidine were infused either together or separately in both high and low dosages into the GM,. Blockades were validated by intra-arterial injection of histamine or the specific agonists betahistine for HI and dimaprit for Hz receptors. The results suggest that the high-dose diphenhydramine produced a nonspecific dilation not seen with the lower dose. Because both the blocked and unblocked vascular beds exhibited the same degree of vasodilation after endotoxin, these studies do not support the hypothesis that endogenously released histamine is responsible for the loss of vascular tone. These studies do verify, however, that a nonneurally mediated loss of skeletal muscle vascular tone is an important factor to consider in the overall cardiovascular hypotension occurring during endotoxin shock. endotoxic shock; lipopolysaccharides; H1 receptors; Hz receptors; diphenhydramine; betahistine; cimetidine; dimaprit; adenosine dilation; skeletal muscle vascular tone; vascular conductance

NUMEROUS PREVIOUSREPORTSfromourlaboratoryand others have shown that a significant loss of skeletal muscle vascular tone accompanies the onset of vascular decompensation during both hemorrhagic (3, 6, 7, 17) and endotoxic shock (1, 4, 5, 8, 9, 17, 18, 28). Indirect evidence suggests that this loss of tone is mediated, at least in part, by prostaglandins (6) of the E series or baroreceptor inhibition as suggested by Koyama et al. (21). However, other vasoactive substances such as histamine, which also mediate vasodilation, have been shown to be released during both hemorrhagic and endotoxin shock (X5,27). The data reported by Cho et al. (13) and Nagy et al. (24) suggest that histamine levels begin to rise in dogs at the onset of hemorrhage and reach peak H882

0363-6135/91

$1.50 Copyright

levels at a time that would be consistent with the onset of skeletal muscle vascular decompensation (i.e., loss of vascular tone). Histamine could interact with H1 receptors to mediate either contraction or relaxation of vascular smooth muscle, whereas Hz receptors mediate only vasodilation in the peripheral vasculature (12, 20). Additionally, there is evidence for the presynaptic existence of both H, (19) and HZ receptors (23, 29), which serve to reduce the amount of norepinephrine released during sympathetic stimulation. These reports all strongly suggest a possible role for histamine acting either directly on histaminergic receptors or indirectly through modulation of the adrenergic nervous system in the sequence of cardiovascular events leading to hypotension induced by either hemorrhage or endotoxin. The primary objective of this study was to determine whether endogenously released histamine acting through the histaminergic receptors in the skeletal muscle vasculature is responsible for the loss of skeletal muscle vascular tone shown to occur during systemic endotoxemia. A secondary objective was to evaluate the relative importance of the H1 and Hz receptors in this response. Because a recent study (9) from the present authors has demonstrated that a greater loss of vascular tone occurred in denervated than in innervated gracilis muscle preparations during endotoxemia, we elected to use only a denervated constant flow, vascularly isolated, double gracilis muscle preparation in which one of the muscles serves as a control for the experimental muscle. The H, and/or HZ receptors were pharmacologically blocked with the use of three different dose levels of diphenhydramine and/or cimetidine, respectively. The level of blockade was evaluated using a close intra-arterial injection of histamine (H, and Ha agonist), betahistine (H, agonist), or dimaprit (Hz agonist). Recirculation of the histamine antagonists to the contralateral control muscle was prevented by collecting and discarding the venous effluent from the treated muscle. METHODS Surgical Procedures Healthy 15 to 20-kg dogs were anesthetized with 30 mg/kg pentobarbital sodium, intubated, and placed in the supine position on a warm-water heating pad. Both the right and left gracilis muscles were vascularly isolated and denervated according to a procedure previously de-

0 1991 the American

Physiological

Society

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HISTAMINERGIC

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IN

scribed (6, 9). When vascular isolation and cannulations were complete, all blood flowing through a perfusion pump also flowed through the gracilis muscles. Vascular isolation was verified by momentarily stopping the pump and noting a fall in the distal perfusion pressures to 15 20 mmHg. The venous circulations from both gracilis muscles were also isolated; and, except when histamine antagonists were infused, the venous outflows returned to the systemic circulation through a common cannula inserted into the left femoral vein. With this in situ preparation, a constant and equal blood flow was maintained to both the control (GM,) and experimental (GM,) gracilis muscles. With pump flow constant, all changes in vascular tone were noted as either an increase or decrease in the pressure perfusing that muscle. Before any of the cannulations were performed, all dogs were primed with 300 U/kg heparin sodium (Sigma) to prevent blood coagulation. A supplemental dose of 150 U/kg was administered after 1 h. To help minimize tissue dehydration, 10 ml/kg sterile Ringer solution with lactate (28 meq/l) was administered intravenously during surgery. A Gould polygraph and Statham P23 Db pressure transducers permitted simultaneous monitoring of four pressures [mean arterial pressure (MAP), central venous pressure (PJ, perfusion pressure to the control muscle (PJ, and perfusion pressure to the experimental muscle (PJ]. The four Gould pressure amplifies were connected to a Dataq Instruments data acquisition system that permitted very precise pressure and time base recordings. On completion of all surgical procedures the perfusion pump flow rate was adjusted to deliver a constant flow rate of 3 ml/min to each muscle. The resulting pump flow rate of 5.5-7.5 mlomin-l. 100 g of gracilis muscle-l has been shown by Duran and Renkin (14) to provide more than sufficient blood flow to maintain the metabolic integrity of resting canine gracilis muscle. Once adjusted, the preparation was allowed 30 min to stabilize. Arterial PO, was maintained X00 mmHg by use of supplemental respiratory oxygen (9). The maximum dilating capability of all vascular beds studied was determined at the end of each experiment by injecting multiple 500-pg doses of adenosine into the arterial perfusion cannulas. This adenosine regime has been shown by this laboratory to provide a short-term maximum loss of vascular tone. Subsequent studies by us have shown that 100 pg of papaverine HCl produces quantitatively the same degree of vasodilation in these skeletal muscle preparations as 500 pg adenosine. Constant

Baroreceptor

Tone

Because the mechanism(s) of action of endotoxin may be on either the peripheral or central portion of the sympathetic nervous system, it was important to compare the vascular responsiveness of gracilis muscles during a constant and controlled baseline level of humoral and sympathetic neural tone. To accomplish this, the systemic arterial blood pressure was held constant at -65 mmHg with the use of a constant pressure reservoir system connected to large-bore cannulas (PE-320) inserted into the aorta through the common carotid arteries. This reservoir provided a constant hydrostatic pressure of 65 mmHg against the animal’s arterial pressure.

MUSCLE

DURING

ENDOTOXEMIA

H883

With the line opened, the animal’s pressure forced blood into the reservoir until the MAP equalized at 65 mmHg. When the systemic arterial pressure fell after endotoxin, the blood in the reservoir automatically flowed back into the animal until the preset pressure was reestablished. This constant arterial pressure technique resulted in a baroreceptor-induced state of both elevated and constant adrenergic tone. A state of low adrenergic neural tone was achieved by section (i.e., decentralization) of the gracilis nerve. Studies by Bond et al. (8) have shown that permanent bilateral common carotid artery ligation results in a 32% increase in gracilis muscle vascular tone lasting at least 4 h and that this elevated tone can be eliminated by section of the gracilis nerve. Because this laboratory has recently shown that the loss of canine gracilis muscle vascular tone induced by systemic endotoxin is greater in denervated than in innervated muscles, all of the present studies were carried out in denervated muscles (9). Protocol Endotoxemia was induced in all animals by intravenous infusion of 2 mg/kg of the lipopolysaccharide derived from Escherichia coli endotoxin (Sigma) in a concentration of 1 mg/ml over a 30-min period. All animals were monitored for an additional 60 min after the completion of the endotoxin infusion. To examine the effect of the histaminergic receptors on the loss of vascular tone after endotoxin, a Harvard double syringe infusion pump was used to simultaneously infuse blocking agents into the GM, and the vehicle (sterile saline) into the GM, arterial perfusion lines. The high doses of the histamine antagonists used in the first three animal groups (i.e., animals with HI-receptor, HZ-receptor, and H, and Hz high-dose receptor blockade) were determined from the earlier reports of Powell and Brody (25, 26) and Camazine et al. (11). The effectiveness of the receptor blockade was evaluated every 15 min by intra-arterial injection of the agonist into both the blocked and unblocked muscles (see Fig. 6). To prevent recirculation of the blocking drugs to the contra1 .ateral control mu scle, all venous blood (3 ml/mi n) from the experimental side was collected and discarded by a method previously described (9). The volume of discarded blood in groups with HI-receptor, HZ-receptor, and H, and HZ high-dose receptor blockade was -70 ml, which was easily made up for by the blood in the pressure-controlling reservoir. HI-receptor blockade. In this group (n =8), 150 mg of diphenhydramine HCl (Sigma) dissolved in 100 ml sterile saline was infused at a rate of 1 ml/min into the GM, while a similar volume of the vehicle was infused simultaneously into the GM,. Considering a blood flow rate of 3 ml/min and the infusion rate of 1 ml/min, the final peripheral blood concentration of diphenhydramine during the 15-min infusion period was -8.6 mM. This 15min diphenhydramine infusion was accomplished during the second 15 min of the endotoxin infusion, and the venous blood from the GM, was collected and discarded during the period 3 min before diphenhydramine infusion until 5 min after the infusion was completed (i.e., a total of 23 min). The HI-receptor blockade was challenged at 0, 15, 30, 45, 60, 75, and 90 min after the initiation of

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H884

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IN

the endotoxin infusion by the intra-arterial injection of 1 pg betahistine (Sigma) in a volume of 0.1 ml into both the experimental and control muscles. &-receptor blockade. In this group (n = 4), 150 mg of cimetidine (Sigma) dissolved in 100 ml sterile saline was infused into the GM, according to the same protocol described in HI-receptor blockade. The final peripheral blood concentration of cimetidine was -10 mM. The HZreceptor blockade was challenged bY the intra-arterial injection of 1 pg dimaprit (S- [3- (NJV-dimethylamino) propyllisothiourea) supplied by SmithKline Beecham in a volume of 0.1 ml into both the control and block muscles. HI and H2 high-dose receptor blockade. In this group of animals (n = 8), 75 mg each of diphenhydramine HCl and cimetidine made up in 100 ml sterile saline were infused into the GM, while the same volume of the vehicle was infused into the contralateral GM,, thus providing peripheral blood concentrations of 4.3 and 5.0 mM for diphenhydramine and cimetidine, respectively. As in the above protocols, the infusion was carried out during the second 15 min of the endotoxin infusion. The resulting histaminergic blockade was challenged by intra-arterial injection of 1 pg histamine (Sigma) in 0.1 ml saline into both the GM, and GM,. HI and Hz low-dose receptor blockade. Because of the observed nonspecific vasodilator effects of high doses of histamine antagonists on vascular smooth muscle tone, a much lower dose of these agents was used in this series (n = 6). In these studies, 29.2 mg of diphenhydramine HCl and 25.2 mg cimetidine were dissolved in 100 ml sterile saline. This solution was infused into the experimental limb at a rate of 0.1 ml/min beginning 15 min before the introduction of endotoxin and extending throughout the entire 90-min observation period (total of 105 min). The final peripheral blood concentration of each of these antagonists was 32.2 PM. The venous blood from the experimental muscle was drained and discarded during the entire 105-min period. Because the volume of discarded blood in this series often exceeded the available blood in the reservoir, it was necessary to replace the discarded blood with an equal volume of dextran 70 (6% wt/vol) in normal saline (Pharmacia). The extent of the histaminergic blockade was challenged by intra-arterial injections of 0.5-1.0 pg histamine made up in a concentration of 10 pg/ml. Statistical Analysis The data plotted in Figs. 2, 4, 7, and 10 represent the mean t SE percentage of maximum vascular conductance (URP,,,) in the blocked and unblocked muscles during and after the 30 min infusion of 2 mg/kg endotoxin. The URP,,,,, (i.e., 100%) was determined by obtaining the minimum perfusion pressures (PPmin) after multiple injections of the vasodilator adenosine at the end of each experiment (see Fig. 6A) and applying the following equation URP,,,

= flow/PPmin - VP

where VP is venous pressure. The dilation ratio (DR) data presented in Figs. 3, 5, 8, and 11 illustrate the effectiveness of the blockade in each of the four groups

MUSCLE

DURING

ENDOTOXEMIA

studied. The DR was calculated by subtracting the minimum perfusion pressure after the injection of the agonist (P2) from the perfusion pressure immediately preceding the agonist injection (PI) and dividing that difference by the preagonist pressure (P,) DR = (P, - PZ)/P1 The DR data presented in Figs. 3, 5, 8, and 11 represent the percentage agonist response in the GM, compared with the GM, (see Fig. 6) v

\

,

%blockade = 100 x DR GM,/DR

GM,

All data generated in these studies were analyzed statistically using analysis of variance (ANOVA) with repeated measures. Differences between particular group means were tested using Student’s t test with the Bonferroni correction for multiple comparisons. A Student’s t test for paired comparisons was used when analyzing data from the same animal. All data are presented as means + SE. Statistical significance was considered to be P c 0.05. RESULTS

Table 1 shows the pre-endotoxin control data expressed as means t SE for the four research groups presented in this study. There were no significant differences in any of the between group control data presented. Note that the control MAPS increased from 121-132 to 156-165 mmHg during bilateral carotid occlusion (BCO) and then were reduced to 66-68 mmHg as a result of the controlled hemorrhage immediately before endotoxin infusion at time 0. The mean control and experimental muscle blood flows ranged between 5.98 and 6.64 ml= min-l -100 ml-‘, which is several times higher than the minimal value of 2.0 ml. min-’ 100 g-l reported by Duran and Renkin (14) to be necessary to maintain the metabolic integrity of resting gracilis muscle in the dog. Figure 1 depicts a typical record showing the MAP, VP, P,, and P, during BCO and hemorrhage. Note the rise in MAP after ligation of the common carotid arteries (BCO), the subsequent transient fall during carotid artery manipulation during cannulation, and the precipitous fall to a plateau between 65 and 70 mmHg during controlled hemorrhage. The fact that the perfusion pressures (PC and P,) did not increase during BCO verifies that the muscles were effectively denervated (8). Both perfusion pressures fell significantly with the onset of hemorrhage, which may be the result of a transient release of histamine (24). The secondary rise in perfusion pressures was probably the result of a hemorrhagic hypotension-induced rise in plasma catecholamines. These pressures then typically decrease over the next 15-20 min before reaching a plateau by 30 min. All animals were allowed to reach this plateau before endotoxin infusion was begun at time 0. l

HI Antagonist In Fig. 2, 2 mg/kg of endotoxin was infused intravenously between 0 and 30 min. During the 15- to 30-min time period, the GM, was exposed to an 8.6 mM concentration of diphenhydramine while an equal volume of

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HISTAMINERGIC

TABLE

RECEPTORS

IN

MUSCLE

DURING

H885

ENDOTOXEMIA

1. Between group control data before intravenous endotoxin and antihistamine administration into dog Control

MAP Group

n

Body Wt

Con

BCO

t=o

Muscle

Wt

&

6.35kO.45 6.04t0.66 6.24k0.42 6.64t0.73

1H 2H 1,2HH

8 4

17.5t0.7

121t6

165~6

66rtl

48.7t3.0

19.3t0.5

12228

160t13

67tl

8

18.6t0.4

124rt6

156tlO

68t1

51.5k5.7 49.5t3.1

1,2HL

6

17.9rt1.2

132t5

163t5

67tl

48.Ok5.3

Exptl URP (x10-q

5.84t0.76 4.57t0.67 5.29t0.97

7.10t0.73

Wt

50.8rt2.9

52.1k6.1 51.3t2.9 49.5t5.6

Muscle &

URP (x1o-2)

5.98t0.35

5.63t0.56 4.80t0.63 5.85t1.1

6.45t0.71

6.99t0.59

6.04t0.33 6.00-+0.70

Values are means t SE; n = no. of dogs. Group 1H dogs were treated with 8.6 mM of H1 blocker diphenhydramine; 2H group was treated with 10 mM of Hz-blocker cimetidine; 1,2HH group received a high dose of both diphenhydramine (4.3 mM) and cimetidine (5 mM); and 1,2HL group received a low dose of both diphenhydramine (32 PM) and cimetidine (32 PM). Body wt in kg; MAP, mean arterial blood pressure (in at time 0; control and- experimental mmHg); Con, MAP before bilateral common carotid artery occlusion (BCO); t = 0, MAP after hemorrhage conductances (URP) calculated by dividing Q by perfusion gracilis muscle wt (in g). Blood flows (Q) expressed as ml 100 g-l min-‘, and vascular pressure (in ml. min-’ 100 8-l. mmHg-l). l

l

l

MAP mmt-u 100 0

I

t BCO

tHEMORRHAGE

5 8

min

-

VP mmw

PC (mmM1

FIG. 1. Polygraph record shows rise in mean arterial pressure (MAP) during common carotid artery occlusion (BCO) and fall in MAP during hemorrhage. Transient fall in MAP after BCO occurred while common carotid arteries were being stretched during cannulation. VP, venous pressure. Note that there was no significant rise in either control (PJ or experimental (P,) perfusion pressure during BCO, indicating an effective denervation. Fall in P, and P, during hemorrhage at arrows may be result of a transient histamine release, whereas secondary rise in these pressures is likely caused by an increased plasma level of vasoconstrictor hormones.

Pe (mmHg1

sterile saline was infused into the GM,. The pre-endotoxin GM, and GM, vascular conductances at time 0 were not significantly different at 19 t 3 and 25 t 3% of maximum, respectively. By 90 min, the saline control conductance increased to 31 t 2% of maximum. The diphenhydramine-treated muscle conductance increased immediately to 57 t 6% at 20 min and gradually fell to 44 -+ 3% by 90 min. The histogram in Fig. 3 shows that after the infusion of diphenhydramine (30-90 min), the DR response to a 1-pg injection of the HI-agonist betahistine maintained an -50% block compared with the contralateral unblocked side (GM,).

duced by systemic endotoxin. The time 0 conductances for four animals were 30 t 10 and 20 t 3% for the GM, and GM,, respectively. At the 90-min point, these percentages rose significantly to 49 t 12 and 41 t 4%; however, these values were not significantly different from each other. Figure 5 shows the effectiveness of the H2 blockade to the specific Hz-agonist dimaprit. Note that even at this very high dose of cimetidine no evidence of an Hz blockade against dimaprit could be demonstrated.

Hz Antagonist

In this series of eight experiments, the peripheral blood concentration perfusing the GM, was maintained at 4.3 mM diphenhydramine and 5.0 mM cimetidine for 15 min while sterile saline was infused into the GM,. Figure 6 is

Data plotted in Fig. 4 show the effect of a 10 mM blood concentration of cimetidine on the vascular dilation in-

High-Dose HI Plus Hz Antagonists

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H886

HISTAMINERGIC

100

*

1

I

I

I

I

0

90

RECEPTORS

I

I

Saline 8.6 mM

l

I

IN

I

MUSCLE

DURING

100

I

I

I

90 /

Diphenhvdramine /

N=8

T

ENDOTOXEMIA I

I

I

I

I

Saline l 10 mM N = 4

I

I

I

I

-

0

//

Cimetidine

60

: I‘!h++++-+ ******* 5 0

40

0

30

:

> X

20 10

IO -I

0

0

Diphenhydramine

1

I

IO

20

Etox

30

-

Infusion

40

I

50

60

I

80

70

I

_

0

90

I

I

I

0

10

20

Time(minutes)

I

I

m

50

I

60

I

I

80

70

90

4. Time/function plot illustrates effect of 10 mM cimetidine on vascular conductance during endotoxemia. Both treated and untreated vascular beds exhibited a loss of vascular tone after endotoxin infusion.

I

I

I

200

1

Diphenhydramine

r

I

I

I

m

180

N=8

I

100

40

Time(minutes)

I

8.6mM

30

FIG.

FIG. 2. Time/function plot illustrates effect of diphenhydramine HCl on vascular conductance during endotoxemia. High dose of diphenhydramine caused a nonspecific increase in vascular conductance. Etox, endotoxin. * P < 0.05.

120

Cimetidine Etox Infusion

N =

I

1 OmM

I

140

s

120

I

I

1

4

160 g cl

I

Cimetidine

T

I

i

1

/

0 *

‘;rcr

100

iz

80

i= 4

60

i5

40

20

20 0

0

0 15

30

45 TIME

60

75

90

(min)

3. Bar graph shows that before infusion of diphenhydramine into GM,, dilation ratios induced by 1 pg betahistine were the same in GM, and GM,. After diphenhydramine infusion, dilation ratio in blocked GM, was significantly reduced. * P < 0.05. FIG.

a typical record that illustrates the perfusion pressure responses to endotoxin, multiple histamine challenges, diphenhydramine plus cimetidine infusion, and the maximum dilator response to adenosine. Note the substantial fall in both control and blocked perfusion pressures with the first histamine injection. This was followed by the beginning of the endotoxin infusion that resulted in an initial rise in both perfusion pressures, suggesting vasoconstriction. Approximately 10 min into the endotoxin

0

15

30

45 TIME

5. Graph shows that cimetidine of the specific Hz-agonist dimaprit. FIG.

60

75

90

(min) failed

to block

a l-pg

injection

infusion, but before the blockers were infused, both pressures began to fall, yet both still responded equally well to histamine. The blockers were added to the experimental infusion line (GM,) at the arrow, which prompted an immediate fall in pressure (nonspecific vasodilation). Additional histamine challenges were given every 15 min with the result that a substantial block was demonstrated throughout the 90-min observation period, even though the overall perfusion pressures continued falling to both the control and blocked vascular beds. Adenosine was

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HISTAMINERGIC

RECEPTORS

IN

MUSCLE

DURING

H887

ENDOTOXEMIA

CONTROL

-

1

BLOCKED

ETOX FIG. 6. Record diphenhydramine

illustrates plus 5 mM

INFUSION

typical perfusion pressure responses to etox, I pg intra-arterial cimetidine infusion (blockers infused), and maximum dilator

added to both perfusion lines at A, showing that even at 90 min postendotoxin a maximal dilation can be obtained. Figure 7 illustrates the effect of these high doses of H1 and H2 antagonists on vascular conductance in response to systemic endotoxin. The 0 time control conductances were 23 t 3% for control and 24 t 4% for the blocked and unblocked muscles, respectively. Introduction of the blockers at 15 min resulted in an immediate increase in GM, conductance to -53% between 20 and 30 min (also seen in Fig. 6). At 90 min, the conductances in the blocked muscles (38 t 3%) were not significantly different than the unblocked controls GM, (39 t 5%), 100

I

I

I 0

90

l

I

I

Saline 4.3 mM 5.0 mM

I

I

I

Diphenhydramine Cimetidine

I

I

although both were significantly higher than their respective pre-endotoxin control values. The bar graph in Fig. 8 shows that from 30 to 90 min a highly significant blockade in the GM, in response to 1 pg histamine compared with the nonblocked GM, can be seen. This histamine blockade is similar in magnitude to the betahistine blockade noted in Fig. 3. Low-Dose

*

J

&

histamine (H), 4.3 mM response to adenosine (A).

/

H, plus ~~ Antagonists

Because the previously described studies all showed some degree of nonspecific vasodilation in response to the antagonists, we elected to conduct a fourth study using low doses of diphenhydramine and cimetidine. In these studies a nerinheral blood concentration of 32.2

1

120

I

T

I

I

1

0

10

20

Diphenhydramine Etox Infuqion

30

40

50

&

60

I

pa

100

N=

20

*

I

4.3mM 5.OmM 8

I

I

I

Diphenhydramine Cimetidine

&

Cimetidine I

I

I

70

80

90

Time(minutes) 7. Illustration shows progressive loss occurs in saline control muscle after systemic nonspecific rise in conductance with onset of cimetidine infusion into experimental vascular 90 min, both blocked and unblocked sides have conductance than they did at 0 time, but they each other. * P < 0.05. FIG.

I

of vascular tone that etox. Note substantial diphenhydramine and bed. Also note that by a significantly higher are not different from

nv 0

15

30

45

60

75

90

TIME (min) 8. Bar graph illustrates that diphenhydramine plus cimetidine significantly blocks vascular response to 1 pug histamine. 0- and 15min challenges were made before infusion of blockers. * P < 0.05. FIG.

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H888

HISTAMINERGIC

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IN

MUSCLE

DISCUSSION

Many investigators have reported that a significant loss of peripheral vascular tone accompanies the systemic hypotension associated with endotoxemia (1, 3, 8, 9, 17, 18, 27, 28). In 1975, Finley et al. (16) found that the muscle blood flow in septic patients was significantly higher than in nonseptic patients. With the use of the rat cremaster muscle, Garrison and Cryer (17) were able to localize this loss of vascular tone to the small arteries. In two recent reports (8, 9) from this laboratory using the same protocol reported here, the following facts were established: 1) the baseline level of vascular tone may not be an important factor when evaluating the systemic effect of endotoxin on peripheral vascular tone (8); 2) intravenous infusion of endotoxin results in a progressive loss of vascular tone in both innervated and denervated gracilis muscle vasculature with the denervated muscle showing twice as much dilation, suggesting a nonneural vasodilating mechanism (9); and 3) infusions of endo100

ENDOTOXEMIA

toxin directly into the arterial perfusion lines failed to result in a loss of tone in either intact or denervated muscle (9). These studies suggest that endotoxin must interact with a systemically dependent mechanism to release a vasodepressor substance that is then transported to the peripheral vasculature, where it causes the loss of vascular tone by a nonneural decompensatory mechanism. One compatible theory proposed by Branemark and Urbaschek (10) more than twenty years ago suggests that endotoxi .ns in .itiate the release of histamine from mast cells that then interact with histaminergic receptors in the vasculature, causing a loss of smooth muscle contractile ability. In 1976, Powell and Brody (25, 26) were able to demonstrate a role for both H1 and Hz receptors in the active reflex vasodilation seen in a dog model. Neither receptor type, however, appeared to be involved with the nonreflex (i.e., nonneural) dilations associated with reactive hyperemia or exercise hyperemia. Also of interest was the report of Levi et al. (22), who suggested that the H, receptors play a greater role in the peripheral vasodilation response to histamine than the Hz receptors. The purpose of the present study was to examine this histamine concept using the canine constant flow perfused double gracilis muscle preparation. This preparation permits the use of one gracilis muscle to serve as an untreated control while the GM, is pretreated with relatively specific HI - (diphenhydramine) and/or HZ-receptor (cimetidine) antagonists. Because we had shown previously that a more dramatic loss of vascular tone was seen in denervated than in innervated preparations (9), we elected to concentrate our current effort on a possible nonneural histaminergic mechanism. Consequently, the peripheral sympathetic neural and humoral influences were neutralized by maintaining carotid sinus pressure constant at 65 mmHg and by gracilis muscle denervation. Figure 1 shows the MAP, VP, P,, and P, responses to BCO and subsequent hemorrhage into the constant pressure reservoir. Note that with the onset of BCO, MAP

PM of each antagonist was maintained continuously from 15 min before endotoxin until the end of the 90-min observation period. Figure 9 shows a record from one of these experiments. Note the initial increase in both the P, and P, that is followed by a gradual but equal fall in both muscles over the 90-min observation period. The vascular conductance data in Fig. 10 show no change in conductances between the onset of blocker infusion at -15 min until the beginning of systemic endotoxin at 0 min. The time 0 conductances are 20 t 2% for control and 23 t 2% for the blocked side. The initial responses to endotoxin are slight falls to 19 t 2 and 20 t 2% that are followed by highly significant increases reaching 46 + 5% for the saline group (GM,) and 54 t 5% for the blocked group (GM,) at 90 min. The bar graph in Fig. 11 shows that this protocol resulted in an effective blockade of the histamine response when compared with the saline control muscle.

MAP (mmW

DURING

-

7

5. _, -

-

---

-

-

--

_

0 7

20 -

VP (mmHg1

10 3-w

FIG. 9. Polygraph record is typical of vascular responses observed when etox was infused systemically. In this study, 32 PM diphenhydramine and 32 PM cimetidine were infused into experimental muscle 15 min before beginning of 30infusion and continued min etox throughout entire 105min observation period. Note initial rise in both P, and P,, which is followed by a progressive and equal fall in both P, and P, over next 90 min.

I-

O

200

I--

Pe 100 (mmHg1 1 0

I TIME (min)

C--

t

t

t

0

15

30

t ETOX

t 60

t 75

t 90

NF"$3N

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HISTAMINERGIC I

I

I

0 *

I

Saline 32pM 32pM

I

RECEPTORS

I

I

I

Diphenhydramine Cimetidine

I

IN

I

I

&

N=6

10 Etox I

0 -20-10

Infusion

I

I

I

I

I

0

10

20

30

40

II

50

60

I

I

70

80

II

90

Time(minutes) 10. Infusion of diphenhydramine plus cimetidine was begun at -15-min point, and infusion continued through 90-min point. Note that blockers did not change 0 time control value compared with saline control, both muscles showed a slight fall in conductance at 10 min into etox infusion, and throughout entire lo- to 90-min period both treated and untreated vascular conductances increased significantly but equally. FIG.

120

I

I

m

-T

100

32pM 32pM

I

I

I

I

Diphenhydramine Cimetidine

&

N=6

/1 iii (3 wx

80

MUSCLE

DURING

i= ii!

*

60

5 i= 4 Q

40

20

0 -15

FIG.

protocol challenge

0

15

30

45

TIME

(min)

60

75

Bar graph verifies that diphenhydramine plus effectively blocked a 0.5pg histamine challenge. was made before infusion of blockers. * P < 0.05.

11.

90

cimetidine -5min

increases by -30% without any increase in either P, or P,, suggesting that the carotid sinus reflex influence on the gracilis vasculature has been effectively interrupted by the prior sectioning of the gracilis nerves (8). The transient dip in MAP after BCO occurred as a result of carotid artery manipulation during their cannulation. Approximately 5 min after BCO, the carotid artery clamps were released, and blood was allowed to flow into

H889

the reservoir (hemorrhage). With these lines left open, blood was free to flow into the reservoir during cardiovascular compensation and back into the animal during MAP at 65-68 decornpensation, thus maintaining mmHg. Note the slight dip in both P, and P, at the arrows. This transient loss of vascular tone after hemorrhage has been attributed by Nagy et al. (24) to be the result of histamine release acting on some nonneural mechanism. This dilation was regularly followed by a secondary increase in both P, and P,, which was likely the result of a hemorrhage-induced increase in plasma catecholamine concentration acting on the postsynaptic al- and extrasynaptic az-adrenoreceptors in the vascular beds. Because of these initial hemorrhage-induced variations in vascular tone, a 30-min stabilization period was provided before the administration of endotoxin. The first two sets of experiments listed below were aimed at differentiating the roles of the H1 and H2 receptors in the peripheral vascular decompensation during endotoxemia. In these studies, high doses of specific antagonists were given to the GM, while the GM,s were infused with an equal volume of the vehicle (sterile saline). The specific agonists betahistine (H,) and dimaprit (Hz) were used to evaluate the extent of the histamine-receptor blockades. In the third group (H1 and Hz high-dose receptor blockade), high doses of both diphenhydramine and cimetidine were administered to the GM, while the extent of blockade was challenged with the nonspecific agonist histamine. The fourth group (H, and Hz low-dose receptor blockade) received much lower doses of diphenhydramine and cimetidine over a period of time that started 15 min before endotoxin infusion began and continued throughout the entire 90 min protocol. As in the group with H, and Hz high-dose receptor blockade, histamine was used to challenge the blockade. Diphenhydramine

0

ENDOTOXEMIA

(8.6 mM)

In the HI-receptor blockade series, the relatively specific HI-receptor antagonist diphenhydramine was infused into the GM, during the second 15 min of the endotoxin infusion. The dose and infusion rate were calculated to provide a peripheral blood concentration of 8.6 mM, which was a dose equivalent to that used by Powell and Brody (25, 26) in 1976. The data presented in Fig. 2 show that with the initiation of the endotoxin infusion the vascular conductance of both GM, and GM, increased slightly by 15 min. However, with the onset of diphenhydramine infusion a far more dramatic increase in GM, conductance resulted that remained above the control muscle throughout the rest of the observation period. Consequently, even though the H, receptors were effectively blocked against the specific agonist betahistine (Fig. 3), the apparent nonspecific dilation at this dose level severely hampered the interpretation of the data. Cimetidine (10 mM) In the Hz-receptor blockade series, the relatively specific Hz-receptor antagonist cimetidine was infused into the GM, at a dose that resulted in a peripheral blood

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Ha90

HISTAMINERGIC

RECEPTORS

IN

concentration of 10 mM. The degree of HZ-receptor blockade was assessed by intra-arterial injection of 1 pg of the relatively specific Hz-receptor agonist dimaprit. Again the GM, that received saline showed a marked dilator response to endotoxin and the GM, showed an apparent nonspecific dilator response to cimetidine (Fig. 3). To further complicate the issue, we were unable to show that even this high dose of cimetidine was ineffective in blocking the Hz-receptor activation response to dimaprit (Fig. 5). This would suggest either that the cimetidine dose was not high enough to block the Hz receptors or that dimaprit is not as selective for the Ha receptors in this vascular bed as was expected, as suggested by Barker and Hough (2). If one assumes that dimaprit is selective for the HZ receptors, the dilator response we obtained with this agent verifies the existence of H, receptors in this vascular bed. In either case, however, the fact remains that the magnitude of dilation at 90 min was the same in the treated and untreated preparations. These results are consistent with those of Levi et al. (ZZ), who reported that the Hz receptors have less of an effect on the peripheral vasculature than the H, receptors. Diphenhydramine

(8.6 mM) Plus Cimetidine

(5 mM)

Figure 6 contains a record of an experiment in which the combination of diphenhydramine (8.6 mM) and cimetidine (5 mM) was infused into GM,. In this study, the starting perfusion pressures were -150 mmHg for the saline-treated control muscle and 130 mmHg for the muscle to be blocked. The first histamine challenge at JY showed a very marked and equivalent fall in perfusion pressures (i.e., vasodilation) to both muscles. Endotoxin was then infused with the result that both perfusion pressures showed transient increases that were probably due to a norepinephrine surge known to follow an endotoxin stress (4). Fifteen minutes into the endotoxin infusion both legs were again challenged with histamine, and again the response was the same in both muscles (also see Fig. 8). At the point indicated in Fig. 6, blocker infusion was initiated with the result that an immediate fall in perfusion pressure was noted in the GM,. We believe this to be a nonspecific dilation due to the high concentrations of these drugs. Note that the control muscle continued to lose pressure (i.e., vasodilation) even though the responses to histamine continued to be significant. The response to histamine in the blocked side showed a significantly smaller response to histamine even though the perfusion pressures were essentially the same as the unblocked GM,. At the end of each experiment, multiple doses of adenosine were injected into the perfusion lines in an effort to determine the maximum vasodilating capacity (i.e., 100% conductance) of each muscle preparation. In this study, both perfusion pressures fell below 25 mmHg. The data plotted in Fig. 7 illustrate the effect that the combined high doses of diphenhydramine and cimetidine had on the endotoxininduced loss of vascular tone. In this series, the 0-min control conductances were 23 and 24% of maximum. At 15 min into the endotoxin infusion, these values had increased to 27 and 28%, respectively. The introduction of treatment at 15 min caused an immediate nonspecific

MUSCLE

DURING

ENDOTOXEMIA

increase in the GM, conductance to 53% at 20 min compared with 30% in the saline-infused muscles. The GM, conductances began to fall after the treatment was stopped at 30 min. By the 90-min point, the salinetreated GM, conductance had increased to 39% and the treated muscles had fallen to essentially the same value (38%). The DR data in response to a 1-pg intra-arterial injection of histamine plotted in Fig. 8 show that the histaminergic-receptor blockade was 40-50% of the control leg at 45, 60, 75, and 90 min. Although the blockade at 30 min was apparently nearly complete at lo%, the nonspecific dilation at this time makes the histamine blockade difficult to interpret. In any event, even though we were able to show a reasonable blockade to histamine at 90 min, the nonblocked GM, and blocked GM, showed the same dilation magnitude in response to endotoxin, suggesting that histamine-receptor activation is probably not responsible for the endotoxin-induced loss of vascular tone. The high histamine antagonist doses used in the first three studies all resulted in nonspecific dilation responses, which made a meaningful data interpretation of the data very difficult. Consequently, we elected to conduct a fourth series of experiments utilizing lower antagonist doses that were effective histamine blockers but did not cause the nonspecific vasodilator response. Diphenhydramine

(32 PM) Plus Cimetidine (32 PM)

Figure 9 shows a record taken from a study in which low doses of diphenhydramine (32 PM) and cimetidine (32 PM) were combined and continuously infused into the GM, from a point 15 min before the initiation of endotoxin infusion until the end of the data collection period at 90 min. As in all studies in this report, the MAP was held constant by use of the blood reservoir connected to the common carotid arteries. The preendotoxin 0-min P, and P, were well matched at -150 mmHg and fell to -75 mmHg at 90 min, which represents a 100% increase in conductance. About 5 min into the endotoxin infusion, both of these perfusion pressures showed a transient increase similar to that seen in Fig. 6. Again we attribute this rise to the known plasma catecholamine surge after endotoxin infusion (4). Figure 10 clearly illustrates the rise in conductances in GM, from 20 t 2% at 0 min to 46 t 5% at 90 min compared with the statistically insignificant difference of 23 t 3 and 54 t 5% for the GM,. The magnitude of this response (130% increase) compares favorably with the 105% increase published previously (9). The statistically insignificant fall to 19 t 2% in GM, and 20 t 2% at 5 min is probably due to the catecholamine surge mentioned above. Note that in this low-dose study no evidence of a drug-induced nonspecific vasodilation was seen. Figure 11 shows that 20-50% of control GM, blockage to histamine was maintained throughout the period of data collection (0 to 90 min). Thus in this study very significant and equivalent lossesin vascular tone (i.e., vascular decompensation) were evident in both the saline control and histamine-blocked sympathetically neutralized gracilis muscles in response to systemically administered endotoxin, suggesting that the decompensation is not the result of an endotoxin-initiated endogenous histaminerelease mechanism.

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HISTAMINERGIC

RECEPTORS

IN

In conclusion, the data presented in this study verify that a non-neurally mediated loss of skeletal muscle vascular conductance is an important factor to consider in the overall cardiovascular hypotension occurring during endotoxin shock. In addition, these studies do not support the hypothesis that endogenously released histamine is responsible for this loss of vascular tone. Previous studies by Bond et al. (9) have shown that endotoxin does not act directly on the vascular smooth muscle to initiate the loss of tone but rather must circulate in the vasculature for up to 10 min before the loss of tone is significant. These results suggest that systemically circulating endotoxin initiates the release of one or more vasoactive substances that act either directly or indirectly on the vascular smooth muscle contractile elements to inhibit the compensatory vasoconstriction expected to occur during arterial hypotension. Appreciation is extended to Dr. Gerald Johnson III for aiding in the statistical evaluation of the data presented in this manuscript. The authors also want to thank Dr. J. Skidmore and SmithKline Beecham for generously supplying the dimaprit used in these studies. This work was supported by a grant-in-aid from the American Heart Association. The National Institutes of Health guidelines for the use of experimental animals were adhered to throughout this study. Address for reprint requests: R. F. Bond, Dept. of Physiology, Univ. of South Carolina, School of Medicine, Columbia, SC 29208. Received

15 October

1990; accepted

in final

form

30 April

MUSCLE

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

1991. 20.

REFERENCES 1. ABEL, F. L., AND R. R. BECK. Canine peripheral vascular response to endotoxin shock at constant cardiac output. Circ. Shock 25: 267274,1988. 2. BARKER, L. A., AND L. B. HOUGH. Selectivity of 4-methylhistamine at H,- and Hz-receptors in the guinea-pig isolated ileum. Br. J. Pharmacol. 80: 65-71, 1983. 3. BOND, R. F. A review of the skin and muscle hemodynamics during hemorrhagic hypotension and shock. Adu. Shock Res. 8: 53-70, 1982. 4. BOND, R. F. Peripheral vascular adrenergic depression during hypotension induced by E. coli endotoxin. Adu. Shock Res. 9: 157169,1983. 5. BOND, R. F. Peripheral circulatory responses to endotoxin. In: Handbook of Endotoxin, Pathophysiology of Endotoxin, edited by L. B. Hinshaw. Amsterdam: Elsevier, 1985, vol. 2, p. 36-75. 6. BOND, R. F., C. H. BOND, L. C. PEISSNER, AND E. S. MANNING. Prostaglandin modulation of adrenergic vascular control during hemorrhagic shock. Am. J. Physiol. 241 (Heart Circ. Physiol. 10): H85-H90,1981. BOND, R. F., AND G. JOHNSON III. Vascular adrenergic interactions during hemorrhagic shock. Federation Proc. 44: 281-289, 1985. BOND, R. F., C. G. SCOTT, J. C. CLEVENGER, C. H. BOND, AND F. L. ABEL. Effects of systemic endotoxin on skeletal muscle vascular conductance during high and low adrenergic tone. Circ. Shock 30: 311-322,199O. BOND, R. F., C. G. SCOTT, L. H. KRECH, AND C. H. BOND. Systemic and local effects of endotoxin on canine gracilis muscle vascular

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conductance. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H498H506, 1990. BRANEMARK, P. I., AND B. URBASCHEK. Endotoxins in tissue injury: vital microscopic studies on the effect of endotoxin from E. coZi on the microcirculation. Angiology 18: 667-671, 1967. CAMAZINE, B., R. P. SHONNON, J. L. GUERREO, R. M. GRAHAM, AND W. J. POWELL, JR. Neurogenic histaminergic vasodilation in canine skeletal muscle: mediation by al-adrenoreceptor stimulation. Circ. Res. 63: 871-883, 1988. CHARBON, G. A., H. A. BROUWERS, AND A. SALA. Histamine H,and HZ-receptors in the gastro-intestinal circulation. Arch. Phurmucol. 312: 123-129, 1980. CHO, Y. W., J. THEGARAJ, D. M. AVIADO, AND S. BELLET. Studies of the myocardial oxidative enzymes during histaminergic shock. Arch. Int. Phurmucodyn. 158: 314-323, 1965. DURAN, W. N., AND E. M. RENKIN. Oxygen consumption and blood flow in resting mammalian skeletal muscle. Am. J. Physiol. 226: 173-177,1974. EMERSON, T. E., JR. Release of vascular effects of histamine, serotonin, angiotensin II and renin following endotoxin. In: Hundbook of Endotoxin, Puthophysiology of Endotoxin, edited by L. B. Hinshaw. Amsterdam: Elsevier, 1985, vol. 2, p. 173-202. FINLEY, R. J., J. H. DUFF, R. L. HOLIDAY, D. JONES, AND J. B. MARCHUK. Capillary muscle blood flow in human sepsis. Surgery 78: 87-94, 1975. GARRISON, R. N., AND H. M. CRYER. Role of the microcirculation to the skeletal muscle during shock. In: Perspectives in Shock Research: Metabolism, Immunology, Mediators, and Models, edited by J. C. Passmore, S. M. Reichard, D. G. Reynolds, and D. L. Traber. New York: Liss, 1989, p. 43-52. GILBERT, R. P. Mechanisms of the hemodynamic effects of endotoxin. Physiol. Reu. 40: 245-279, 1960. KIMURA, T., AND S. SATCH. Inhibition of cardiac sympathetic neurotransmission by histamine in the dog is mediated by HIreceptors. Br. J. Phurmucol. 78: 733-738, 1983. KOO, A. In vivo characterization of histamine HI- and HZ-receptors in the rat stomach microcirculation. Br. J. Phurmucol. 78: 181189, 1983. KOYAMA, S. Baroreflex participation of cardiovascular response to E. coZi endotoxin. Jpn. J. Physiol. 36: 267-275, 1986. LEVI, R., D. A. OWEN, AND J. TRZECIAKOWSKI. Action of histamine on the heart and vasculature. In: Pharmacology of Histamine Receptors, edited by Ganellin and Parson. Bristol, UK: Wright, 1982, p. 236-297. MCGRATH, M. A., AND J. T. SHEPHERD. Inhibition of adrenergic neurotransmission in canine vascular smooth muscle by histamine. Circ. Res. 39: 566-573, 1976. NAGY, S., A. NAGY, A. ADAMICZA, I. SZABO, K. TARNOKY, AND A. TRAUB. Histamine level changes in the plasma and tissues in hemorrhagic shock. Circ. Shock 18: 227-239, 1986. POWELL, J. R., AND M. J. BRODY. Identification and specific blockade of two receptors for histamine in the cardiovascular system. J. Phurmucol. Exp. Ther. 196: l-14, 1976. POWELL, J. R., AND M. J. BRODY. Participation of H, and HZ histamine receptors in physiological vasodilator responses. Am. J. Physiol. 231: 1002-1009, 1976. VICK, J. A., B. MEHLMAN, AND M. HEIFFER. Early histamine release and death due to endotoxin. Proc. Sot. Exp. BioZ. Med. 137: 902-906,197l. WEIL, M. D., D. L. MACLEAN, M. B. VISSCHER, AND W. W. SPINK. Studies on the circulatory changes in the dog produced by endotoxin from gram-negative microorganisms. J. CLin. Inuest. 35: 11911198,1956. WESTFALL, T. C. Neuroeffector mechanisms. Annu. Reu. Physiol. 42: 383-397, 1980.

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