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Cardiovascular Research 2002 56(2):225-234; doi:10.1016/S0008-6363(02)00543-6
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Sepsis causes presynaptic histamine H3 and {alpha}2-adrenergic dysfunction in canine myocardium

Zao-Qin Cheng, Deepak Bose, Han Jacobs, R Bruce Light and Steven N Mink*

University of Mannitoba, Departments of Medicine, Pharmacology and Therapeutics, Anesthesiology, and Biochemistry and Molecular Biology, Winnipeg, Manitoba, Canada

minksn{at}cc.umanitoba.ca

* Corresponding author. MD Health Sciences Center, GF-221 700 William Ave Winnipeg, Manitoba R3E-0Z3, Canada. Tel.: +1-204-787-2914; fax: +1-204-787-1932.

Received 4 April 2002; accepted 25 June 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Histamine H3 receptors and {alpha}2-adrenoceptors are presynaptic receptors that modulate norepinephrine (NE) release from sympathetic nerves innervating the cardiovascular system. We previously showed that cardiac H3 receptors are activated in sepsis, and that this activation leads to a decrease in the adrenergic response (AR) [J. Appl. Physiol. 85 (1998) 1693-1701] H3-receptors and {alpha}2-receptors appear to be coupled to GTP binding regulatory proteins (G) that modulate transmitter release by reducing calcium current into the nerve terminals through neuronal calcium channels. There may also be interaction between H3-receptors and {alpha}2-receptors on AR that may occur either at the receptor or a more downstream level. Methods: In the present study, we examined the effect of septic plasma on AR in a canine ventricular preparation in which field stimulation was used to produce AR. We determined whether there was interaction between H3-receptors and {alpha}2-adrenoceptors and tested whether H3 activation would attenuate the {alpha}2-agonist and {alpha}2-antagonist effects of clonidine and yohimbine, respectively. We also determined whether the mechanism by which septic plasma decreases the adrenergic response involves inactivation of an inhibitory G protein and used pertussis toxin (PTX) to assess this effect. Results: We found that septic plasma attenuated AR produced by field stimulation, and that this decrease was mediated by a PTX sensitive inhibitory G protein. H3 activation also attenuated the {alpha}2-agonist and {alpha}2-antagonist effects on adrenergic activation as compared with nonseptic plasma. Conclusion: We conclude that presynaptic sympathetic dysfunction may contribute to cardiovascular collapse in sepsis.

KEYWORDS Adrenergic (ant)agonists; Autonomic nervous system; Contractile function; G-proteins, Septic shock


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Myocardial dysfunction is an important contributor to the development of cardiovascular collapse in septic shock [1]. Among the physiologic compensatory mechanisms that would potentially limit this loss of myocardial contractility, stimulation of the cardiac sympathetic neurons leading to an increase in the neuronal release of norepinephrine (NE) would be expected to be important. However, even under normal conditions, the factors that regulate the release of NE from the cardiac sympathetic neuron are complex. The release of NE is influenced by activation of presynaptic receptors that are located close to the axon terminals of neurons [2,3]. Presynaptic receptors are mechanisms through which the receptors to a neuron’s own transmitter (autoreceptor) or a different mediator (heteroreceptor) modify transmitter release evoked during depolarization of the neuron [2–4].

In the heart, among the many presynaptic receptors that have been identified, histamine H3 (H3) and alpha2-receptors ({alpha}2) appear important in the regulation of the endogenous release of NE during neural activation [4–6]. The {alpha}2-receptor is an autoreceptor, such that the endogenous release of NE during stimulation causes an inhibition of further release of this mediator. An {alpha}2-receptor agonist, such as clonidine therefore attenuates the release of NE during neural stimulation, while the opposite situation occurs with the {alpha}2-antagonist yohimbine.

Under normal conditions, H3 receptors are quiescent, since there is little circulating histamine to bind to this receptor [4,7]. However, in pathological conditions associated with enhanced adrenergic activity, enough histamine is released to activate these receptors. The distribution of histamine in the heart parallels with the known distribution of NE, and high concentrations of histamine are present in sympathetic nerves [4,7]. The amount of histamine required for H3 activation is quite small (10–9–10–7 M) as compared with that needed for H1 and H2 activation (10–5 M) [4,8,9]. In an in vivo model of sepsis, we previously showed that concentrations of plasma histamine reached after 4 h of bacteremia were sufficient to cause H3 activation, and that this activation contributed to the cardiac depression observed [8]. Moreover, in a ventricular trabecular preparation, we showed that a plasma fraction [≤30 000 molecular weight (Mw)] from septic animals caused a decrease in the neural adrenergic response, and that this decrease was antagonized by histamine H3 receptor blockade [8]. These results suggest that H3 receptor activation may be an important component contributing to cardiac adrenergic dysfunction in sepsis.

Interaction between the H3 receptor and {alpha}2-adrenoceptor has been found in some organ systems, but not in others [10,11]. In cortex slices of the rat brain, Schlicker et al. [10] found that H3 receptor activation inhibited the increase in adrenergic response ordinarily observed when {alpha}2-antagonists were added to the preparation. Likewise, in mouse brain cortex, H3 receptor activation attenuated the reduction in the agonist response observed when {alpha}2-adrenoceptor agonists were added to the preparation [10]. These findings indicate negative cooperativity between receptors. On the other hand, Poli et al. [11] could not detect an interaction between the H3 receptor and {alpha}2-adrenoceptors in guinea pig duodenum.

In terms of signal transduction, cardiac H3 and {alpha}2 presynaptic receptors are both thought to be coupled to GTP binding regulatory proteins (G protein) that are found on the cell membrane and serve as transducers to convey signals to their effector proteins [2,12]. G proteins display stimulatory and inhibitory properties, and among other mechanisms may modulate intracellular adenyl cyclase and inward calcium currents [2]. Possibly, activation of H3 and {alpha}2-agonist receptors increase inhibitory activity of G proteins (Gi) that in turn may decrease the adrenergic response in sepsis. Further, it has been shown that pertussis toxin (PTX), which inactivates inhibitory G proteins, can be observed to attenuate H3 receptor activation in the heart [4,13]. Thus, if septic plasma were to cause H3 activation by coupling to an inhibitory G protein, then this effect could be blocked by PTX.

In the present study, we examined the interaction between the H3-receptor and {alpha}2-receptor under septic conditions in canine ventricular trabeculae. We used field stimulation to produce the adrenergic response (AR) [4,8,14]. The objectives were to determine whether there is negative or positive cooperativity between the H3 receptor and {alpha}2-adrenoceptor in limiting AR in sepsis, and to determine whether septic plasma affects the cardiac AR by means of coupling to a PTX sensitive inhibitory G protein.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Overall protocol
This study was approved by the Central Animal Care Committee at the University of Manitoba. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

In Part 1 of this protocol, two sets of experiments were performed. In Part 1A, septic plasma fraction (see below) was harvested from an in vivo preparation. As previously described, septic plasma fraction has been shown to reduce the adrenergic response in ventricular trabeculae [8]. After septic plasma fraction was added to the trabecular preparation, the effects of the addition of the {alpha}2-antagonist yohimbine, and the {alpha}2-agonist clonidine were determined in respective groups of experiments. In Part 1B, histamine was added to the trabecular preparation to cause H3 receptor activation, after which the adrenergic response with the {alpha}2-antagonist yohimbine, and the {alpha}2-agonist clonidine, were again determined in respective experiments. The results were compared in the preparations exposed to septic plasma fraction or histamine to see if findings were similar. In the histamine experiments, the concentration used was 10–7 M. This concentration approximated that found in 0.5 ml of pooled septic plasma of ten animals when the sample was placed into the 5-ml tissue bath (see preparation below). In Part 2, the ventricular trabecular preparation was treated with PTX in order to determine whether PTX blocks the effect of septic plasma fraction on AR (see below).

2.2 In vivo preparation
The procedure to obtain septic plasma fraction was identical to that previously described [15,16]. In brief, dogs (20–30 kg) were anesthetized with pentobarbital sodium (30 mg/kg), intubated with an endotracheal tube, and ventilated (15 ml/kg; Harvard Apparatus variable speed ventilator, Model 607, South Natick, MA, USA) to maintain pCO2 and pH at {approx}35 mmHg and 7.35, respectively. The dogs received supplemental oxygen to maintain pO2>100 mmHg. After 1 h of stability, sepsis was induced by intravenous infusion of 1010 colony-forming units (CFU) of live E. coli (0111:B4). The bacteria were suspended in normal saline and given over a period of 1 h. Approximately 5x109 CFU/h of E coli were then infused for 3 additional hours [15,16].

A 30-ml volume of plasma was obtained from the animal before sepsis and after 4 h of E coli infusion. The 4-h period was chosen because cardiac depression and hypotension have been universally observed to occur at this time in this model [15]. The respective nonseptic and postseptic samples were passed through a 30 000 molecular weight filter to isolate the ≤30 000 Mw fraction. This fraction was used because histamine is found in this fraction, and also because, this fraction has been shown to contain sepsis associated cardiac depressant activity that occurs independently of abnormalities found in sympathetic neural stimulation [15,16]. Thus, this fraction would be expected to simulate the changes in cardiac function observed in sepsis.

2.3 In vitro preparation
The ventricular trabecular preparation used in this protocol was identical to that previously described [15,16]. Mongrel dogs (3–10 kg) were anesthetized with pentobarbital sodium. The hearts were removed, flushed with 50 ml of cold Krebs–Henseleit solution (KH), and placed in ice-cold oxygenated KH. Thin trabeculae (<1 mm diameter) were obtained from the right ventricle and tied at each end with 6-O silk thread. Each muscle was suspended in a vertical constant temperature bath (5 ml) containing KH (in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.4 KH2PO4, 25 NaHCO3, and 11 dextrose). The solution was gassed with 95% O2–5% CO2, and was maintained at 37 °C. Isometric contractions at optimum length were recorded with a force transducer (model FTO3C, Grass Instruments). The muscle was stimulated electrically at 0.5 Hz via platinum punctate bipolar electrodes with trains of four rectangular pulses (2 ms duration) at an intensity of 100% greater than threshold.

As described in a previous study [8], sympathetic nerve stimulation was produced by increasing the pulse width of the electrical stimulus trains from 2 to 20 ms, keeping other stimulus parameters unchanged. After a train of stimuli that caused sympathetic field stimulation was performed (of which the number could be varied until a plateau in response was observed), the pulse width was reduced to 2 ms to restore control responses. The increase in tension seen with sympathetic nerve stimulation was calculated as a percent increase from basal twitch amplitude. In previous experiments, it was determined that propranolol (10–5 M) completely abolished AR during field stimulation, whereas atropine had no effect [8].

2.4 Experimental protocol
In Part 1A, after baseline measurements of AR were made, 0.5 ml of septic plasma fraction was placed into a tissue bath, while in another bath, 0.5 ml of nonseptic plasma fraction was used. Either yohimbine or clonidine was then added separately to an individual bath. A dose–response relationship was determined, in which each agent was added to the bath to produce sequential concentrations of 10–7, 10–6 and 10–5 M. Measurements were obtained approximately 15 min apart. The concentrations used were based on the work of previous investigators [4,10]. Whenever possible, simultaneous experiments were performed with septic and nonseptic plasma samples, so that the results between the two conditions could be compared with ventricular trabeculae obtained from the same donor dog. This limited the variability in the sympathetic response observed between experiments. However, this approach could not be uniformly followed because it depended on the highly variable number of trabeculae that could be obtained from a given donor dog. Experiments were additionally performed to confirm that the H3 receptor blocker clobenpropit reversed the effect of septic plasma on the adrenergic response [17]. The concentration used was 10–5 M based on the results obtained in a previous experiment [8].

In Part 1B, after baseline recordings were made, histamine (10–7 M) was added to the trabecular bath. This concentration was used to mimic that found in the pooled 0.5 ml plasma fraction sample of ten septic animals when the sample was placed into the 5-ml tissue bath. This concentration has also been shown to decrease in the sympathetic response by activation of H3 receptors [8], but not to activate H1 and H2 receptors [4,8,9]. As described for the septic experiments, after histamine was added to the tissue bath, either yohimbine or clonidine was then added in steps to produce final concentrations of 10–7, 10–6 and 10–5 M. Companion experiments, in which Krebs–Henseleit (KH) solution was added to the bath instead of histamine, were simultaneously performed in most cases, so that the results could be compared with ventricular trabeculae obtained from the same donor dog. In addition, time control experiments were performed to establish the effect of repetitive stimulations on AR over an identical interval. In these experiments, KH solution was added to the bath in volumes identical to those used in the clonidine and yohimbine experiments over the course of the experiment.

In Part 2, ventricular trabeculae were incubated with PTX. This was based on the report by others that PTX inhibited a G sensitive inhibitory protein in the inactive state [4,13]. Ventricular trabeculae were suspended in the organ bath filled with KH. PTX was added to the bath to yield a final concentration of 3.0 µg/ml. After incubation for 45 min, the trabeculae were washed every 30 min with fresh KH solution for a total of 5.25 h. The same protocol was used for the untreated experiments, except that an equal volume of KH was substituted for the toxin.

In one part of the experiments included in Part 2, septic plasma fraction was placed into respective PTX treated and nontreated baths, so that the effect of the PTX on AR could be determined. The volumes of the septic plasma fraction used were 0.15, 0.30 and 0.5 ml. In a second part of Part 2 experiments, histamine at concentrations of 10–7, 10–6 and 10–5 M were placed into the PTX treated and the nontreated preparations in a similar manner. In order to determine whether the effect of PTX on the adrenergic response was a nonspecific one, in a final group of experiments, nonseptic plasma fraction of identical volumes was placed in a PTX treated bath, and its effect on the subsequent AR was determined.

2.5 Statistical analysis
Comparisons between the treatment and nontreatment groups were made by split plot ANOVA in which the changes observed between conditions were compared. Within group comparisons were performed by one-way repeated measures ANOVA. A Student–Newman–Keuls’ (SNK) multiple-comparison test was used when multiple comparisons were made. In the analysis, the number of experiments used (n) in a subgroup of experiments is identical to the number of animals studied. Two to four muscles could usually be obtained from a given animal. Each muscle was then used in a different subset of experiments. The results are expressed as mean±1S.E.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
An example of the active tension tracings obtained from a series of experiments results found when nonseptic plasma fraction was added to the trabecular bath is shown in Fig. 1. Baseline tracings are shown in the upper left hand panel in which the trabecula is stimulated at 0.5 Hz and isometric tension is recorded as active tension. At the time indicated by the first arrow, neural stimulation was initiated. Initially, a slight decrease in active tension developed because of a change in the pathway by which depolarization proceeds along the trabecular muscle (see Ref. [14] and legend). After this transient decrease in tension, active tension was gradually augmented. This effect is due to an increase in the release of norepinephrine that can be blocked by propranolol as described in our earlier study [8]. The addition of nonseptic plasma to the bath caused little change in active tension (middle panel). When yohimbine was subsequently added to the trabecular bath, there was a large increase in isometric tension. In the lower panels, the results obtained with clonidine are shown. Clonidine, which causes a decrease in norepinephrine release, resulted in a decrease in active tension as compared with the two previous conditions.


Figure 1
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  		Fig. 1 Isometric tension measured in the ventricular trabecular preparation is plotted against time for the nonseptic plasma fraction. Adrenergic response is indicated by interval between arrows. The small dip in isometric tension at beginning of stimulation is believed to represent the effect of synchronization as described by Blinks [14]. This may be due to abnormal conduction of action potential when sympathetic stimulation is initially applied and then stopped. Horizontal dashed line represents basal tension. Nonseptic plasma fraction caused little change in the adrenergic response (middle panel). The {alpha}2-antagonist yohimbine (upper right hand panel) caused an increase in adrenergic response, while the {alpha}2-agonist clonidine (lower right hand panel) caused a decrease in response as compared with respective previous conditions.

 
An example of the tracings obtained when septic plasma fraction was added to the bath is shown in Fig. 2. Septic plasma fraction caused a decrease in the sympathetic response as compared with the baseline condition. In the septic condition, the AR found when yohimbine (upper panel) and clonidine (lower panel) were added to the bath are quantitatively less than respective responses found in the nonseptic condition.


Figure 2
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  		Fig. 2 Isometric tension measured in the ventricular trabecular preparation is plotted against time for the septic plasma fraction (see legend to Fig. 1). Septic plasma fraction (middle panels) caused a decrease in adrenergic response as compared with baseline. The {alpha}2-antagonist yohimbine (upper right hand panel) caused little increase in adrenergic response, while the {alpha}2-agonist clonidine (lower right hand panel) caused little decrease in response as compared with respective previous conditions.

 
In Part 1A, the mean AR obtained with yohimbine at the different concentrations are shown in Table 1. In Fig. 3, AR are expressed as the difference in response ({Delta}AR%) between subsequent conditions. Baseline AR were not different among the groups. Although there was a large variability in the baseline adrenergic response found among the experiments, this variability did not affect the directional changes in the response when the different agents were added to the preparation. When septic plasma fraction was added to the bath (upper panel in Fig. 3), the mean AR decreased by –16% as compared with baseline. As compared to the immediately preceding condition, the changes with yohimbine were relatively small. In contrast, when nonseptic plasma fraction was added to the bath, the mean AR decreased only by –3%. As compared to the immediately preceding condition, the mean changes observed when yohimbine was added to the trabecular bath were quite large. These changes were significantly different from the septic plasma experiments (note, also, that in some cases {Delta}AR% as calculated from the mean of the differences found in Table 1 may be slightly different from the results obtained when the average {Delta}AR% is calculated from the individual differences as shown in Fig. 3. This discrepancy is a reflection of the degree to which each of the individual differences mirrors that of the mean difference. The greater the variance then the greater the difference).


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  		Table 1 Effects of the {alpha}2-antagonist yohimbine and {alpha}2-agonist clonidine on percent adrenergic response in sepsis (mean±S.E.)a

 

Figure 3
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  		Fig. 3 Mean (±S.E.) changes measured in the per cent adrenergic response between the different conditions are shown for the {alpha}2-antagonist yohimbine (upper panel) and the {alpha}2-agonist clonidine (lower panel) under septic and nonseptic conditions in Part 1A. The changes measured were between (Treatment–Baseline), (10–7 M concentration–Treatment), (10–6M–10–7M), and (10–5–10–6M). n=number of animals studied (one muscle was used from each animal). Statistics included split plot ANOVA and SNK. +P<0.05 between groups.

 
The mean AR obtained with clonidine are shown in Table 1. In Fig. 3 (lower panel), AR are expressed as the difference in response ({Delta}AR%) between subsequent conditions. When septic plasma fraction was added to the bath, the mean AR decreased by –21% as compared with baseline. Relative to the immediately preceding condition, the changes observed when clonidine was added in the presence of septic plasma fraction were relatively small compared to those found in the presence of nonseptic plasma fraction.

In other experiments, it was found the decrease the AR brought about by septic plasma fraction was completely reversed by H3 receptor blockade with clobenpropit. The mean AR (±S.E.) were 30±6% at baseline, 10±4%* after septic plasma fraction, and 30±5%+ after H3 receptor blockade (n=12; *, P<0.05 vs. baseline; +, P<0.05 vs. septic plasma fraction). On the other hand, there was no effect of H3 receptor blockade on AR after nonseptic plasma fraction was added to the preparation, and the mean results (n=13) were 28±6% at baseline, 28±6% after nonseptic plasma fraction, and 33±11% after H3 receptor blockade.

In Part 1B, the mean results observed with histamine are shown in Table 2; the changes calculated between conditions are displayed in Fig. 4. The findings were similar to those observed in the sepsis experiments. In Fig. 4 (upper panel), histamine at 10–7 M caused a decrease AR that averaged –23% below baseline. The addition of yohimbine at the three doses brought about little increase in AR as compared with the preceding condition. In contrast, when KH was added as a control, the average change in AR was only 2% as compared with baseline; the addition of yohimbine significantly increased the size of the adrenergically mediated response as compared with the preceding measurements.


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  		Table 2 Effects of the {alpha}2-antagonist yohimbine and {alpha}2-agonist clonidine on percent adrenergic response in histamine experiments (mean±S.E.)

 

Figure 4
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  		Fig. 4 Mean (±S.E.) changes in the per cent adrenergic response measured between the different conditions are shown for the {alpha}2-antagonist yohimbine (upper panel) and the {alpha}2-agonist clonidine (lower panel) under histamine and placebo (i.e. KH) conditions in Part 1B. The changes measured were between (Treatment–Baseline), (10–7 M concentration–Treatment), (10–6–10–7 M), and (10–5–10–6 M). Statistics included split plot ANOVA and SNK. +, P<0.05 between groups.

 
The changes observed when clonidine was added to the preparation after histamine are depicted in Fig. 4 (lower panel). Histamine caused a decline in AR of –21% as compared with baseline, while the subsequent addition of clonidine at the three concentrations used brought about little increase in the sympathetic response. In contrast, when KH was added to the trabecular preparation, the average change in AR was only 1% as compared with baseline. The subsequent addition of clonidine to the bath significantly decreased AR as compared with the preceding measurements.

Time control experiments were also performed and showed no effect of repetitive stimulations on AR over the time internals studied [(n=8) 67±8, 76±12, 84±12, 78±9 and 83±8%].

In Part 2, the effect of PTX on the adrenergic response was examined when septic plasma fraction (Part 2A) (Fig. 5, upper panel) or histamine (Part 2B) (Fig. 5, lower panel) was added to the organ bath in respective experiments. PTX inhibited the decline in AR observed under these conditions. On the other hand, as compared with baseline, there was no effect of PTX on AR when nonseptic plasma fraction at volumes of 0.15, 0.30 and 0.50 ml was added (n=7); AR averaged 20±5, 21±5, 21±5 and 20±4%, respectively.


Figure 5
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  		Fig. 5 Mean (±S.E.) adrenergic response after aliquots of septic plasma fraction in Part 2A (upper panel) and histamine in Part 2B (lower panel) were placed into the bath of pertussis toxin treated and nontreated ventricular trabeculae. Statistics by split plot ANOVA and SNK. *, P<0.05 vs. proceeding condition; +, P<0.05 between treated and nontreated muscles.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present study shows that septic plasma attenuated AR produced by field stimulation of the sympathetic nerves in isolated canine ventricular trabeculae. This occurred as a result of H3 receptor activation, and this decrease was most likely mediated by G protein signaling. Endou et al. [4] found that prejunctional histamine H3 receptors modulated the depolarization-dependent release of transmitter from sympathetic nerve endings in guinea pig atria, and that these receptors were coupled to a PTX sensitive inhibitory G protein. Our results not only support this finding, but show that inhibition by G protein signaling is a mechanism by which depression in AR may occur in sepsis.

Furthermore, the present study shows that after receptor activation is produced by septic plasma fraction, subsequent modulation of AR by the {alpha}2-agonist clonidine or the antagonist yohimbine is attenuated. As shown in Fig. 3 (upper panel), in the absence of septic plasma fraction, the {alpha}2-antagonist yohimbine at a final concentration of 10–5 M produced a net increase in AR of {approx}60% (i.e. the summation of all responses) as compared with baseline, a finding which is consistent with the known action of this agent on increasing the concentration of NE available for augmenting contractility. However, in the presence of septic plasma fraction, AR with yohimbine increased only minimally above baseline values (Fig. 6).


Figure 6
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  		Fig. 6 Schematic showing the effect of presynaptic function in the absence (left hand panel) and presence of histamine (H3) receptor activation (right hand panel). In the absence of H3 receptor activation, yohimbine caused a marked increase in norepinephrine release (NE) and adrenergic response (AR), while clonidine caused decreases in these parameters. Histamine H3 receptor activation (right hand panel) caused a decrease in NE release and AR; the responses to clonidine and yohimbine were attenuated compared with the findings observed in the absence of H3 activation.

 
Similarly, in the presence of nonseptic fraction or KH, the {alpha}2-agonist clonidine at a concentration of 10–5 M caused a net decrease in AR of approximately –30% below the baseline value (Figs. 3 and 4Go lower panels), this effect being consistent with the known action of this agent on decreasing the concentration of NE available for contractility. In contrast, the effect of clonidine was near zero under conditions of septic plasma fraction or histamine. These results suggest negative cooperativity between the cardiac H3 receptor and the {alpha}2 receptor in this sepsis model. Once activation has been achieved by septic plasma, AR is locked in a fixed state and was not receptive to further modification by the {alpha}2-agonist and {alpha}2-antagonist agents (see Fig. 6).

How could this interaction occur? Schlicker et al. [10] suggested that the mutual interaction observed between the {alpha}2-autoreceptor and the H3-heteroreceptor may occur at the level of the receptors themselves or interaction at a common step beyond the receptors was possible as well. Allgaier et al. [18] proposed that the interaction between the {alpha}2-autoreceptor and various heteroceptors might occur at the level of the G proteins. It has been shown that presynaptic receptors are associated with and promote activation of G proteins by stimulating release of GDP bound to the guanine nucleotide-binding site of the {alpha}-subunit of the protein [12,19]. Release of GDP is followed by GTP binding, causing dissociation of the heterotrimer into derivative substrate {alpha} and β{gamma}. Hydrolysis of bound GTP to GDP allows the {alpha} subunit to be reassociated with the β{gamma}-dimer. Both the {alpha} and β{gamma} subunits can regulate G protein coupled-effectors in a selective manner that can be independent, synergistic, or antagonistic [19]. Thus, there exist many points along the regulatory pathway of G protein signaling where interaction between H3 and {alpha}2 receptor might occur thereby modifying AR in sepsis.

The structural and functional classification of G proteins has further been defined by the {alpha} subunits [12,19]. The G{alpha}i class, of which there are many subgroups, contain a conserved COOH terminal cysteine residue that is the site of ADP ribosylation catalyzed by pertussis toxin. Blockade of cellular responses to stimulation by pertussis toxin treatment has been shown to be an effective experimental procedure employed to implicate this class of G{alpha} subunits in specific cellular signaling processes [12,19]. Accordingly, our results are consistent with the hypothesis that the decline in the AR observed in sepsis results from activation of an inhibitory G protein mediated by activation of the H3 receptor.

The mechanism by which activation of an inhibitory G protein causes a subsequent decrease in AR in sepsis may be related to its effect on intracellular calcium ion concentrations. Both the L and N type calcium ion channels of neurons are regulated by G proteins [20]. Endou et al. [5] found that H3 receptor signaling in the heart reflect involvement of an inhibitory G protein that could be eliminated by blockade of NB-type calcium channels. In guinea pig duodenum, Poli et al. [11] found that both H3 receptor activation and the inhibitory effect induced by {alpha}2-adrenoceptor activation in electrically stimulated duodenum was closely associated with a restriction of calcium ions. Thus, {alpha}2-receptors as well as H3-receptors may modulate transmitter release by reducing calcium current into the nerve terminals through neuronal calcium channels [4,20].

Although our results cannot establish whether mutual interaction between the {alpha}2-autoreceptor and the H3-heteroreceptor occurred at the level of the receptor, or at a more downstream pathway, it may be useful to consider some of the possible explanations for the present findings. In Part 1, activation of H3 receptors by septic plasma fraction or histamine caused a reduction in AR of about –20% in both the clonidine and yohimbine experiments, and the subsequent addition of the {alpha}2-antagonist yohimbine or the {alpha}2-agonist clonidine had only a minimal effect on AR. Thus, under these conditions, the combined effects of activation of the two presynaptic receptors on the adrenergic response did not appear to reflect a simple algebraic summation of the two components. In the case of yohimbine, under nonseptic conditions, the net increase in AR observed after all of the doses of yohimbine were added was {approx}60% (i.e. the net summation of changes observed in Fig. 3). Under septic conditions, the average decrease in response observed with septic plasma fraction as compared with baseline was –20%. Thus, if the agents acted in an additive manner, then one would have expected a net 40% increase (i.e. 60–20=40%) when both agents were combined. In contrast, when all the changes were added in Fig. 3 (upper), the net response with yohimbine was approximately zero as compared with baseline. Similarly, under nonseptic conditions, the net decrease in response observed with clonidine alone was –30%. In the septic experiments, one would have expected a –50% decrease [i.e. (–30%)+(–20%)=–50%] in AR as compared with baseline, if the responses were additive. On the other hand, the net effect observed with septic plasma fraction was {approx}–20%.

One explanation for these findings is that after H3 receptor activation had occurred, the {alpha}2-receptor was altered, so that it was unresponsive to further activation or blockade. An alternative explanation is that interaction occurred at the level of the G protein. After septic plasma caused H3 receptor activation, calcium current was reduced through the neuronal channels by coupling to an inhibitory G protein and the closing of calcium ion channels. Since the neuronal calcium channels are blocked to further signaling, both the {alpha}2-agonist and {alpha}2-antagonist agents would have little effect on the adrenergic response. Therefore, competing H3 and {alpha}2 receptor activation may modulate the rate and extent to which G protein effectors may be invoked at any point along this biochemical pathway leading to the interaction observed in the present study.

In summary, the central finding of the present study is that septic plasma applied to canine ventricular trabeculae attenuates AR produced by neural sympathetic stimulation by H3 receptor activation. Within the limitations of this approach, this decrease was mediated by a PTX sensitive inhibitory G protein. The results obtained with septic plasma fraction and histamine were the same. Although these findings suggest that histamine may be the sole cause of the decrease in AR in sepsis, it is not possible to exclude the possibility that other circulating factors may have contributed to the effect observed with septic plasma fraction. The present study also shows that there is mutual interaction between the H3 receptor and {alpha}2 receptor in sepsis, although our results do not elucidate the mechanism by which this crosstalk occurs. The interpretation of events related to interaction is complicated by the fact that the factors that regulate G protein are complex and not completely understood. Although more research is needed to investigate this aspect, the present study suggests that presynaptic receptor dysfunction contributes to the cardiovascular collapse observed in sepsis.

Time for primary review 26 days.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Manitoba Heart and Stroke Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Parker M, Shelhamer J, Bacharach S, et al. Profound but reversible myocardial depression in patients with septic shock. Ann Intern Med (1984) 100:483–490.[Abstract/Free Full Text]
  2. Schlicker E, Kathmann M, Detzner M, Exner H.J, Gothert M. H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions. G protein and adenylate cyclase. Arch Pharmacol (1994) 350:34–41.
  3. Starke K, Gothert M, Kilbinger H. Modulation of neurotransmitter release by presynaptic autoreceptors. Physiolog. Rev. (1989) 69:864–989.[Free Full Text]
  4. Endou M, Poli E, Levi R. Histamine H3 receptor signaling in the heart: possible involvement of Gi/Go proteins and N-type Ca2+ channels. J Pharmacol Exp Ther (1993) 269:221–229.[Web of Science]
  5. Imamura M, Poli E, Omonyi A.T, Levi R. Unmasking of activated histamine H3 receptors in myocardial ischemia: their role as regulators of exocytotic norepinephrine release. J Pharmacol Exp Ther (1994) 271:1259–1266.[Abstract/Free Full Text]
  6. Ishikawa S, Sperelakis N. A novel class (H3) of histamine receptors on perivascular nerve terminals. Nature (1982) 327:158–160.
  7. Gross S, Guo Z, Levi R, Bailey W.H, Chenouda A.A. Release of histamine by sympathetic nerve stimulation in the guinea pig heart and modulation of adrenergic responses. Circ Res (1984) 54:1210–1215.
  8. Li X, Eschun G, Bose D, et al. Histamine H3 activation depresses cardiac function in experimental sepsis. J Appl Physiol (1998) 85:1693–1701.[Abstract/Free Full Text]
  9. Guo Z, Levi R, Graver L, Robertson D, Gay W.A. Inotropic effects of histamine in human myocardium: differentiation between positive and negative components. J Cardiovasc Pharmacol (1984) 6:1210–1215.[Web of Science][Medline]
  10. Schlicker E, Behling A, Lummen G, Malinowska B, Gothert M. Mutual interaction of histamine H3 receptors and {alpha}2-adrenergic terminals in mouse and rate brain cortex. Naunyn-Schmiedeberg’s Arch Pharmacol. (1992) 345:639–646.[CrossRef][Web of Science][Medline]
  11. Poli E, Pozzoli C, Coruzzi G, Bertaccini G. Signal transducing mechanism coupled to histamine H3 receptors and {alpha}2-adrenoceptors in the guinea pig duodenum: possible involvement of N-type Ca2+ channels. J Pharmacol Exp Ther (1994) 270:788–794.[Abstract/Free Full Text]
  12. Morris J, Malbon C.C. Physiological regulation of G protein-linked signaling. Physiol Rev (1999) 79:1373–1430.[Abstract/Free Full Text]
  13. Nozaki M, Sperelakis N. Pertussis toxin effects on transmitter release from perivascular nerve terminals. Am J. Physiol (1989) 256(Heart Circ Physiol 25):H455–H459.[Web of Science][Medline]
  14. Blinks J.R. Field stimulation as a means of effecting the graded release of autonomic transmitters in isolated heart muscle. J Pharmacol Exp Ther (1966) 151:221–235.[Abstract/Free Full Text]
  15. Gomez A, Wang R, Unruh H, et al. Hemofiltration reverses left ventricular dysfunction during sepsis in dogs. Anesthesiology (1990) 75:671–685.[CrossRef]
  16. Jha P, Jacobs H, Bose D, et al. Effects of E. coli sepsis and myocardial depressant factor on interval–force relations in dog ventricle. Am. J. Physiol. (1993) 264(Heart Circ. Physiol. 33):H1402–H1410.[Web of Science][Medline]
  17. Kathmann M, Schlicker E, Metzner M, Timmerman H. Nordimaprit, homodiaprit, clobenpropit, and imetit: affinities for H3 binding sites and potencies in a functional H3 receptor model. Arch Pharmacol (1993) 348:498–503.
  18. Allgaier C, Herting G, von Kugelgen O. The adenosine receptor-mediated inhibition of noradrenaline release possibly involves a N-protein and is increased by {alpha}2-autoreceptor blockade. Br J Pharmacol (1987) 90:403–412.[Web of Science][Medline]
  19. Neer E.J, Clapham D.E. Roles of G protein subunits in transmembrane signalling. Nature (1988) 333:129–134.[CrossRef][Medline]
  20. Clapham D.E. Direct G protein activation of ion channels. Annu Rev. Neurosci. (1994) 17:441–446.[CrossRef][Web of Science][Medline]

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S. N. Mink, Z.-Q. Cheng, R. Bose, H. Jacobs, K. Kasian, D. E. Roberts, L. E. Santos-Martinez, and R. B. Light
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