Propofol causes hypotension which is mediated by both direct vasodilation and changes in sympathetic output. It has been demonstrated that propofol possesses direct vasodilator properties in vitro [1,2]. Other investigators, as well as ourselves, have found that inhibition of calcium influx in vascular smooth muscle cells is one of the mechanisms by which propofol causes vasodilation [3,4]. Decreased outflow from the sympathetic nervous system also contributes to decreased vascular resistance. This is indicated by the attenuation of baroreflexes [5-7]. Further support for a sympatholytic effect comes from an investigation of changes in renal sympathetic nerve activity which found that propofol produced parallel decreases in blood pressure, heart rate, and renal sympathetic nerve activity via central nervous system effects . We hypothesized that, in addition to attenuation of central sympathetic output, inhibition of sympathetic neurotransmitter release at the perivascular nerve terminals is also a potential contributor to the decreased vascular resistance.
The present in vitro investigation was designed to examine the effects of clinically relevant concentrations of propofol on the response to electrical field stimulation which is mediated by norepinephrine release from the sympathetic perivascular nerve terminal. We compared the effects of propofol on this neurally mediated response to the nonneurally mediated response to exogenous norepinephrine in the rat femoral artery in vitro.
Animals were managed in accordance with the guidelines of the Canadian Council on Animal Care (Ottawa, Canada). Male Sprague-Dawley rats (250-400 g) were killed by stunning and cervical dislocation. The femoral arteries were exposed, removed, and placed in cold Krebs' solution, and fat and connective tissue were dissected away from the vessels under a microscope. The vessels were cut into transverse ring segments 4 mm in length and mounted in a double-walled, temperature-controlled organ bath with internal volume of 5 mL containing Krebs' solution (composition, in mM: NaCl, 118; KCl, 4.72; CaCl2, 2.52; MgSO4.7H2 O, 0.78; KH2 PO (4), 1.2; glucose, 5.6; NaHCO3, 19), aerated with 95% O2-5% CO2 at 37 degrees C. The vascular ring segments were placed around two stainless steel wires (diameter 204 micro meter) which were mounted in stainless steel holders. One holder was fixed to a FT03 force-displacement transducer (Grass Instrument Co., Quincy, MA) which was attached to a Grass Model 7 polygraph to record changes in vessel wall tension. The other holder was fixed to a Marzhauser MM3 micromanipulator (Fine Science Tools, North Vancouver, British Columbia, Canada) which was used to apply passive tension to the vessels.
Vessels were mounted at minimal tension to prevent rotation and damage to the endothelium and allowed to equilibrate for at least 1 h before experiments were started, at which time a passive tension of 750 mg was applied.
To examine the release of endogenous norepinephrine via electrical field stimulation of perivascular nerves, two parallel platinum electrodes were inserted into the organ bath so that they ran along either side of the vessel. These electrodes were connected to a Grass Model S4 stimulator; at the source, the stimulus had a duration of 0.5 ms and a supramaximum voltage of 80 V.
Responses to exogenous norepinephrine were generated in the form of noncumulative norepinephrine concentration response curves as follows: exogenous norepinephrine was added to the organ bath, the contractile response was allowed to reach a plateau, the norepinephrine was washed out of the bath, and tension was allowed to return to baseline before the next test dose was added. Five-minute intervals were allowed between doses. Final bath concentrations (micro Meter) were 0.01, 0.02, 0.05, 0.1, 0.2, 0.5. When successive concentration-response curves were performed in the same tissue, a 30-min interval was allowed between successive trials.
Responses to endogenous norepinephrine were generated using the electrical stimulation apparatus described above. Electrical field stimulation frequency-response curves were generated by stimulating the vessel for 20-s pulse trains every 5 min at frequencies of 1, 2, 5, 10, 20, and 50 Hz. To confirm that the response to electrical field stimulation was mediated by released noradrenaline, a frequency-response curve was generated in the absence and presence of the alpha-adrenoceptor antagonist phentolamine (10-6 M).
To verify the reproducibility of the electrical field stimulation frequency-response curves and to account for any neurotransmitter depletion, three sequential curves were performed in the absence of any pharmacologic manipulation. Controls for repetition of the frequency-response curve in the same tissue consisted of three consecutive frequency-response curves performed both with and without norepinephrine administration for repletion. Repletion was performed as follows: norepinephrine (5 times 10-7 M) was administered between frequency response curves to replenish any depletion of perivascular nerve norepinephrine stores. The use of this technique to replenish neurotransmitter stores was based on studies by Urabe et al. .
To examine the effects of propofol on the responses to exogenous norepinephrine and endogenous norepinephrine released via electrical field stimulation, norepinephrine concentration-response curves and frequency-response curves were generated while tissues were bathed in Krebs' solution containing propofol. Separate experiments were done using each of the following final concentrations of propofol: 0.5, 1.0, 2.0, 5.0, and 10.0 micro gram/mL.
Propofol is formulated as an emulsion in a vehicle which is identical in composition to Intralipid Registered Trademark (Kabivitrum Canada Ltd., Newmarket, Ontario, Canada; 10% soya bean oil, 1.2% egg phosphatide, and 2.25% glycerol). To examine any effects of this vehicle, norepinephrine concentration-response curves and frequency-response curves were performed in Krebs' solution containing concentrations of Intralipid Registered Trademark that corresponded to the amount of Intralipid Registered Trademark in each of the test concentrations of propofol.
The protocol was performed in the order shown here: Equation 1 Responses to exogenous norepinephrine in A, B, and C were expressed as a percentage of the maximum response to exogenous norepinephrine in A, the first norepinephrine concentration-response curve performed (control). Responses in subsequent norepinephrine concentration response-curves were statistically compared using the Friedman nonparametric analysis of variance for repeated measures followed by Dunn's post-hoc test.
Responses to endogenous norepinephrine in A, B, and C were expressed as a percentage of the response to 20 Hz electrical field stimulation in A, the first frequency response curve performed (control). Statistical analyses were performed as for noncumulative norepinephrine concentration-response curves.
To compare the responses to exogenous and endogenous norepinephrine, the responses to 0.05 micro Meter norepinephrine and 5 Hz electrical field stimulation (the responses which best approximated 50% of the maximum response to each agonist) and the responses to 0.5 micro Meter norepinephrine and 20 Hz electrical field stimulation (the responses which best approximated the maximum response to each agonist), in the presence of Intralipid and propofol, were expressed as a percentage of the control responses at those concentrations and frequencies. The response to 0.05 micro Meter was compared to the response to 5 Hz electrical field stimulation, and the response to 0.5 micro Meter norepinephrine was compared to the response to 20 Hz using the Mann-Whitney nonparametric U-test.
Norepinephrine ((-)Arterenol, bitartrate salt) was purchased from Sigma Chemical Company (St. Louis, MO). Phentolamine (regitine mesylate) and all components of Krebs' solution were purchased from BDH Chemicals Ltd. (Mississauga, Ontario).
Propofol was obtained in the form of Diprivan Registered Trademark from ICI Pharmaceuticals Group (ICI Americas, Inc., Wilmington, DE), which is 10 mg/mL propofol emulsified in soybean oil (100 mg/mL), glycerol (22.5 mg/mL), and egg lecithin (12 mg/mL). Intralipid Registered Trademark (soybean oil 100 mg/mL, glycerol 22.5 mg/mL, and egg lecithin 12 mg/mL) was obtained from KabiVitrum Canada Inc. (Newmarket, Ontario).
Using 10-6 M phentolamine, which was capable of abolishing the response to norepinephrine (10-8 to 5 times 10-7 M) (data not shown), the noradrenergic nature of the response to electrical field stimulation was tested Figure 1. The response to electrical field stimulation was abolished completely at frequencies from 1 to 5 Hz, and attenuated by 80%-97% at higher frequencies, indicating that the majority of the response was due to released norepinephrine.
The response to exogenous norepinephrine remained constant with three repetitions of the noncumulative norepinephrine concentration response curve (data not shown), allowing for comparison of these curves in the presence of Intralipid Registered Trademark and propofol.
When the frequency response curve was repeated three times in succession, the response to electrical field stimulation tended to decrease in successive trials Figure 2a, presumably due to depletion of the endogenous norepinephrine neurotransmitter. The third repetition resulted in statistically significant differences from the first trial at 10 and 50 Hz. When norepinephrine (0.5 micro Meter) was added to the bath for 1-2 min between successive frequency response curves, there were no significant differences between successive trials Figure 2b, so this repletion was performed in subsequent experiments to allow for comparison of successive frequency-response curves in the presence and absence of drugs.
Propofol attenuated the response of rat femoral artery to exogenous norepinephrine Figure 3, a-e. No statistically significant attenuation of the response to norepinephrine was seen in the presence of the lowest concentration used, 0.5 micro gram/mL propofol Figure 3a, but statistically significant attenuation of the response to norepinephrine was seen with 1.0 micro gram/mL propofol at 0.05-0.5 micro Meter norepinephrine Figure 3b, with 2.0 micro gram/mL propofol at 0.5 micro Meter norepinephrine Figure 3c, with 5.0 micro gram/mL propofol at 0.2 and 0.5 micro Meter norepinephrine Figure 3d, and with 10.0 micro gram/mL propofol at 0.1-0.5 micro Meter norepinephrine Figure 3e.
The response to endogenous norepinephrine released via electrical field stimulation was decreased by propofol Figure 4, a-e. Once again, the lowest concentration of propofol used (0.5 micro gram/mL) did not reveal any statistically significant differences Figure 4a, but significant differences were seen with 1.0 micro gram/mL at 1-20 Hz Figure 4b, with 2.0 micro gram/mL at all frequencies (1-50 Hz) Figure 4c, with 5.0 micro gram/mL at 2-50 Hz Figure 4d, and with 10 micro gram/mL at all frequencies Figure 4e.
The vehicle, Intralipid Registered Trademark, showed a tendency to attenuate the response to exogenous norepinephrine, but there were no statistically significant differences between the response to norepinephrine in the presence of Intralipid Registered Trademark and the control response Figure 5a.
Like the response to exogenous norepinephrine, the response to endogenous norepinephrine tended to show a slight decrease in the presence of Intralipid Registered Trademark, but no statistically significant differences were observed Figure 5b.
The points that best approximated 50% of the maximum responses to exogenous norepinephrine and endogenous norepinephrine released via electrical field stimulation occurred at 0.05 micro Meter norepinephrine and 5 Hz electrical field stimulation Figure 6 a and b. The points that best approximated the maxima occurred at 0.5 micro Meter norepinephrine and 20 Hz electrical field stimulation Figure 7 a and b. These points were used to compare the relative effects of propofol on the responses to exogenous norepinephrine and electrical field stimulation.
There was no significant difference between the effects of Intralipid Registered Trademark on the responses to exogenous norepinephrine and electrical field stimulation Figure 6a, Figure 7a. The response to electrical field stimulation was attenuated significantly more than the response to exogenous norepinephrine by 2.0, 5.0, and 10.0 micro gram/mL propofol Figure 6b at 50% of the maximum response. The response to electrical field stimulation was attenuated significantly more than the response to exogenous norepinephrine by 5.0 and 10.0 micro gram/mL propofol Figure 7b at the maximum.
This study attempted to determine whether propofol has presynaptic effects on sympathetic nerve terminals in addition to dilating vascular smooth muscle directly. Comparisons of the response to electrical field stimulation, which elicits contraction in vascular smooth muscle by stimulating sympathetic perivascular nerves to release endogenous norepinephrine, and the response to exogenous norepinephrine, which causes vasoconstriction by a direct action on vascular smooth muscle Figure 3 and Figure 4 demonstrate that propofol inhibits the responses to both exogenous and endogenous norepinephrine released from perivascular nerves by electrical field stimulation in the in vitro rat femoral artery.
To demonstrate that norepinephrine was the most appropriate exogenous drug with which to compare the response to electrical field stimulation, the nature of the response to electrical field stimulation was investigated. Urabe et al.  demonstrated that the contractile response to electrical field stimulation of the rat femoral artery is neurally mediated, since it is abolished by tetrodotoxin, and that the nerves mediating the response are sympathetic, since guanethidine also abolished the response. We confirmed the noradrenergic nature of the response using the nonspecific alpha-adrenoceptor antagonist, phentolamine Figure 1. Phentolamine (10-6 M, a concentration that completely inhibited the response to exogenous norepinephrine) abolished the response to electrical field stimulation at frequencies up to 5 Hz, and substantially reduced responses to frequencies from 10 to 50 Hz. It was therefore concluded that the response to electrical field stimulation in the isolated rat femoral artery was almost entirely due to endogenous, neurally released norepinephrine, and that exogenous norepinephrine was the best single drug with which to compare the response to electrical field stimulation.
Comparison of differential effects of drugs on the vascular response to exogenous norepinephrine versus the response to electrical field stimulation has been used previously to investigate presynaptic effects of drugs. A comparison similar to that presented here was used by Stadnicka et al.  in their investigation of the vascular effects of several inhalational anesthetics. In the present study, Figure 6b and Figure 7b show that the response to electrical field stimulation is inhibited by propofol to a greater extent than can be accounted for by the inhibition of the smooth muscle response to norepinephrine alone. This supports the hypothesis that the release of norepinephrine, as well as the tissue responsiveness to the agonist, is inhibited by propofol. The attenuation of the response to electrical stimulation cannot be accounted for by the effect of propofol on the postsynaptic response to norepinephrine alone, and almost certainly signifies an additional, presynaptic effect on norepinephrine release.
Our conclusion, that propofol attenuates the release of norepinephrine from perivascular nerve terminals, is supported by the more indirect study of Deegan et al.  who showed that spillover of neurally released norepinephrine into plasma is decreased in the presence of propofol. Additional support for this conclusion would be provided by studies measuring the release of endogenous norepinephrine from nerve terminals directly.
The responses to both norepinephrine and electrical field stimulation tend to be diminished by exposure to Intralipid Registered Trademark Figure 5. This trend is not statistically significant, but it cannot be ruled out that Intralipid Registered Trademark may make a contribution to the inhibition of these responses by propofol. However, as shown in Figure 6a and Figure 7a, no difference was seen between the effects of Intralipid Registered Trademark on the response to norepinephrine versus the response to electrical field stimulation at either point. Therefore, if Intralipid Registered Trademark contributed to the depression of the response to norepinephrine and electrical field stimulation, its role could be accounted for entirely by a postsynaptic effect on the smooth muscle. Thus, the additional inhibition of the response to electrical field stimulation at more than the inhibition of the direct response to norepinephrine Figure 6b and Figure 7b is consistent with an additional presynaptic action of propofol.
A confounding factor, limiting the interpretations that can be made from the data presented here, is the difference in the receptors mediating the vasoconstriction elicited by exogenous norepinephrine versus electrical field stimulation. Although both responses are mediated by norepinephrine, they interact with different receptor populations. The rat femoral artery expresses both the alpha1- and alpha2-adrenoceptor subtypes postsynaptically, both of which would contribute to the response to exogenous norepinephrine. However, the response to sympathetic nerve stimulation in the rat femoral vascular bed is elicited by stimulation of alpha1-adrenoceptors only . This is not true of all vascular beds; the rat saphenous bed  and human cutaneous vessels  both have innervated alpha1- and alpha2-adrenoceptors. Considering the different receptor types mediating the neurogenic and nonneurogenic responses, an alternative explanation for the observation that propofol inhibits neurogenic contractions more than exogenous norepinephrine-elicited contractions would be a more selective inhibition of alpha1-adrenoceptors versus alpha2-adrenoceptors. However, the study by Chang and Davis  showed that propofol did not inhibit the contractile response produced by added calcium in rat aorta exposed to phenylephrine, an alpha1-adrenoceptor agonist, in calcium-free solution. Thus, selective alpha1-adrenoceptor inhibition is not implicated as a mechanism for propofol's effect on vascular smooth muscle.
In summary, our results support the hypothesis that decreased sympathetic output via inhibition of norepinephrine release from perivascular nerve terminals is a potential contributor to the decreased vascular resistance and resultant hypotension that is caused by propofol.
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