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Correction for Daggubati et al., Cardiovasc Res 36 (2) 246-255.
Cardiovascular Research 1999 44(1):176-184; doi:10.1016/S0008-6363(99)00174-1
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Angiotensin II modulates conducted vasoconstriction to norepinephrine and local electrical stimulation in rat mesenteric arterioles

Finn Gustafsson* and Niels-Henrik Holstein-Rathlou

Department of Medical Physiology, Division of Renal and Cardiovascular Research, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

* Corresponding author. Tel.: +45-3532-7404; fax: +45-3532-7418 finng{at}mfi.ku.dk

Received 1 March 1999; accepted 28 April 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Objective: Localized application of a vasoconstricting agent onto the wall of an arteriole results not only in a local constriction of the vessel, but also in a conducted vasoconstriction which is detectable more than a millimeter upstream and downstream from the application site. We investigated the effect of intravenous infusion of angiotensin II (ANG II), losartan or methoxamine on conducted vasoconstriction to local application of norepinephrine (NE) or local electrical stimulation onto the surface of rat mesenteric arterioles in vivo. Methods: In anesthetized male Wistar rats (n=43) NE (0.1 mM) or a local depolarizing current was continuously applied onto mesenteric arterioles using micropipettes. Local and conducted vasoconstriction was measured using videomicroscopy. Conducted responses were measured 200–1000 µm upstream from the application site. Results: Systemic infusion of ANG II (4 ng/min) raised mean arterial blood pressure by 6±2 mm Hg and increased the conducted but not the local vasoconstrictor response to NE (P<0.02). Infusion of the {alpha}1-agonist methoxamine raised blood pressure to the same extent, but did not change conducted vasoconstriction significantly. Blockade of endogenous ANG II by infusion of the AT1-receptor blocker losartan decreased conducted vasoconstriction to NE (P<0.03). In parallel with the findings using NE, ANG II increased (P<0.05) and losartan decreased (P<0.01) conducted vasoconstriction when local electrical stimulation was used to initiate the conducted vascular response. Conclusion: The findings suggest that conducted vasoconstriction to NE and local electrical stimulation in rat mesenteric arterioles are modulated by ANG II, an increase in the plasma levels of ANG II increasing conducted vasoconstriction.

KEYWORDS Experimental; Vasculature; Circulatory physiology; Gap junctions; Microcirculation; Renin angiotensin system


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Localized application of a vasoconstricting agent onto the wall of an arteriole results not only in a local constriction of the vessel, but also in a conducted vasoconstriction which is detectable more than a millimeter upstream and downstream from the application site. Conducted vasomotor responses are of importance for the coordination of microvascular blood flow control and tissue perfusion [1]. The mechanism behind conducted vasomotor responses has not been fully resolved, but cell-to-cell coupling via endothelial- or smooth muscle cell gap junctions appears to play a central role in the response [2]. Conducted vasomotor responses resulting from external application of vasoactive substances onto arterioles have been studied in several tissues including hamster cheek pouch and striated muscle and rat kidney and cerebrum [3–6]. In the rat mesentery, propagation of vasomotor responses has been described in arterioles only after intraluminal injection of norepinephrine (NE) [7], and after external application of epinephrine and NE onto capillaries [8]. No information is available on conducted vasomotor responses after external microapplication of vasoconstrictors onto arterioles in this vascular bed.

Modulation of conducted vasomotor responses in arteriolar networks could be of physiological importance in controlling tissue perfusion. However, studies on potential modulating effects by physiologically relevant factors on conducted vasomotor responses are very few in number. Angiotensin II (ANG II) has been shown to modulate cell-to-cell coupling in isolated pairs of cardiomyocytes [9] and in ventricular trabeculae and papillary muscle fibers [10]. Since ANG II is known to have widespread effects on the microcirculation, we hypothesized that ANG II might also alter the conducted vasomotor responses in arterioles. Therefore, using an in vivo preparation of rat mesenteric arterioles, the aims of the present study were: (1) To describe the characteristics of conducted vasomotor responses to local vasoconstrictor stimuli by both application of NE and local electrical stimulation, and (2) to determine whether conducted vasoconstriction could be modulated by perturbations of the renin–angiotensin system by either elevation of plasma ANG II through infusion of exogenous ANG II or by blockade of endogenous ANG II through administration of the angiotensin AT1-receptor antagonist losartan.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusion
 References
 
2.1 Animal preparation
Male Wistar rats (the Panum Institute, Copenhagen, Denmark) (n=58) weighing 215–325 g were used for the experiments. The experimental protocol was approved by the National Research Animal Committee. Anesthesia was induced in a chamber containing 5% halothane in a 35%/65% O2/N2-mixture followed by administration of 2% halothane in a 35%/65% O2/N2-mixture on a mask. Two polyethylene catheters (PE-10) were placed in the left jugular vein for infusions, and a catheter (PE-50) was inserted in the right carotid artery for continuous measurement of blood pressure (TBM4, World Precision Instruments, Aston, United Kingdom). Finally, a tracheostomy was performed to ensure nonobstructed airways. After the initial surgical procedure halothane anesthesia was replaced by intravenous infusion of pentobarbital (120–150 µg/min). The animal was placed on a servocontrolled heated table maintaining body temperature at 37°C. Following a median laparotomy, a loop (4–5 cm) of the small intestine was exteriorized and placed on a small stage slightly elevated from the table carrying the animal. A hole in the stage covered with a 40x24-mm covering-glass allowed transillumination and microscopic observation of the mesentery. The exteriorized intestine and mesentery were superfused with a 37°C physiological saline solution (0.9%) at a rate of 2–3 ml/min.

2.2 In vivo observation of mesenteric arterioles
The mesenteric vasculature was observed using a 20x-water immersion objective with long working distance (UMPlanFI, Olympus, Tokyo, Japan) mounted on an upright microscope (BX50WI, Olympus). The field was viewed on a monitor (Trinitron, PWM 1442 QM, Sony, Tokyo, Japan) using a monochrome CCD camera (CCD 72S, Dage-MTI, Michigan City, IN), and recorded on videotape for off-line analysis. The final magnification of the image was {approx}700x and the spatial resolution was {approx}0.5 µm. The microscope was mounted on a motorized moveable stage (Micromanipulator Mini 25, Luigs and Neumann, Ratingen, Germany). The controller of the three stage motors was connected to a personal computer enabling storage and retrieval of microscope positions along the course of a vessel.

2.3 Application of norepinephrine
Glass pipettes were pulled to an outer tip diameter of approximately 8 µm using a micropipette puller (P-87, Sutter Instruments, Novato, CA) and backfilled with a 0.1 mM solution of NE (Arterenol, Sigma Chemicals) to which lissamine green (5%) had been added. The micropipette was placed in a microperfusion pump (Hampel, Neu Isenburg, Germany) mounted on a micromanipulator (Leitz, Wetzler, Germany). The tip of the micropipette was positioned perpendicular to the arteriole 2–3 µm from the vessel wall.

By adjusting the delivery rate of NE we aimed at inducing a 30–50% reduction in resting internal diameter at the stimulation site. The delivery rate necessary to achieve this constriction ranged from 1–10 nl/min. After a stable local reaction had been achieved, the delivery rate of NE was kept constant during the measurements of local and upstream responses (up to 1 mm from the pipette) (Fig. 1). After completion of a recording of conducted vasoconstrictor responses the microscope was returned to the application site to ensure that the magnitude of the local response was unchanged. Three experiments were discarded because the local response had changed more than 10% during the recording of the upstream responses. In eight experiments application of NE induced neither a local nor a conducted response. In one experiment a conducted but not a local response was observed after application of NE. These experiments were not included in the analysis. Therefore, the total number of experiments (rats) used for the final data analysis in the norepinephrine stimulation series were 31.


Figure 1
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  		Fig. 1 Local and conducted vasoconstriction induced by norepinephrine released from pipette (P). Upper panel: control conditions at the pipette (A) and 600 µm upstream from the pipette (indicated by an arrow) (B). Lower panel: local (C) and conducted vasoconstriction (D) 600 µm from the pipette (indicated by an arrow). L: lymphatic vessel. Bar in upper left corner represents 20 µm.

 
To avoid convection and to minimize upstream diffusion of NE, the superfusate was directed downstream and parallel to the vessel under examination. Due to the addition of dye to the NE solution it was possible to assure that upstream convection of NE did not occur during experiments.

2.4 Local electrical stimulation
In addition, vessel contraction was induced by applying a current directly onto the arteriolar wall using the technique recently described by Steinhausen et al. [11]. In brief, pipettes identical to those used in the norepinephrine series were backfilled with a 2 M NaCl solution and connected to the negative pole of an isolation unit (Iso-Flex, AMPI, Israel) via a Ag–AgCl wire. Pipette resistance ranged from 1–2 M{Omega}. A platinum wire placed in the tissue bath served as a reference electrode. The isolation unit was controlled by a Grass stimulator (S44, Grass Medical Instruments, Quincy, MA) to produce a continuous train of unipolar pulses with a frequency of 10 Hz, pulse width of 2 ms, and voltages between –20 and –50 V. The tip of the pipette was placed as close to the vessel wall as possible and voltage was adjusted to produce a 30 – 50% reduction in the local internal diameter. In each experiment the voltage used before and after infusion of ANG II or losartan was identical.

2.5 Experimental protocol
Experiments were started after the preparation had stabilized for approximately 30 min. An intravenous infusion of saline (0.9%) at 20 µl/min was begun at the start of the equilibration period and continued throughout the experiment. Only one vessel was examined in each animal. Five series of experiments were performed. In two series local and conducted vasoconstrictor responses to local application of NE (n=13) or local electrical stimulation (n=6) were measured before and after 15 min of intravenous infusion of ANG II (4 ng/min, Sigma Chemicals). In one series (n=10) local and conducted vasoconstrictor responses to NE were measured before and after 15 min of intravenous infusion of methoxamine (5 µg/min, Glaxo-Wellcome). Finally, in two series local and conducted vasomotor responses to NE (n=8) or local electrical stimulation (n=6) were measured before and 15 min after an intravenous bolus injection of 3 mg losartan (Merck Research Laboratories) dissolved in 0.5 ml 0.9% saline. In all experiments the pipette was kept in an identical position and local and conducted responses were measured at identical sites of the arteriole before and after administration of ANG II, methoxamine, or losartan.

2.6 Control experiments
A series of control experiments were carried out in arterioles that had proven reactive to stimulation with NE in the concentration and delivery rates used in the experimental protocol. Microapplication of lissamine green (5%) added to physiological saline elicited neither local nor conducted responses (n=3). To test whether diffusion or convection of NE contributed to the conducted vasoconstrictor response, NE (0.1 mM) at delivery rates of up to 20 nl/min was applied at distances of 40, 50 and 100 µm from an arteriole (n=2). Neither a local nor a remote vasoconstriction was induced by this procedure. When the tip of the pipette was repositioned immediately adjacent to the vessel, ejection of NE caused both a local and a conducted vasoconstriction. Repeated applications of NE (separated by a 5-min interval) were performed in nine vessels. In all vessels, both the local and the conducted responses were highly reproducible over time.

The local and conducted responses to electrical stimulation were abolished by slight retraction (5–10 µm) of the pipette from the arteriolar wall (n=5). Repositioning the pipette adjacent to vessel wall reinduced local and remote vasoconstriction. Repeated stimulation up to five times interspaced by a 3-min interval yielded identical local and conducted responses for each voltage used (–20 to –60 V) (n=2).

2.7 Data acquisition and analysis
Off-line analysis of the recorded experiments was performed using manual tracking of endothelial edges at the sites of interest. In the experiments using NE as the vasoconstricting agent internal vessel diameters were measured locally and at distances of 200, 400, 600, 800 and 1000 µm upstream from the stimulation site. In the experimental series using local electrical stimulation vascular diameters were only measured locally and at an upstream site located 600 µm from the tip of the pipette. The response at each site was expressed as the absolute change in the internal diameter.

2.8 Statistics
All values are given as means±SE. Baseline arteriolar diameters, blood pressure changes and NE application rates in the ANG II-, methoxamine-, and losartan groups were compared using Student’s t-test for paired and unpaired data or analysis of variance (ANOVA) as appropriate. In the experimental series using NE for stimulation, conducted responses (change in internal diameter) at sites from 200 µm to 1000 µm upstream from the application site were compared by ANOVA for repeated measures [12]. The analysis was performed via a multiple linear regression approach [13]. If the ANOVA showed a significant effect of the infused agent (the treatment factor) on the remote vasoconstrictions, responses at individual sites were compared using the Least Significant Difference (LSD) test for post hoc comparisons [12]. For experiments where electrical stimulation was used, local and conducted responses (600 µm) were compared using Student’s t-test for paired data. A P-value <0.05 was considered significant.

In the experimental series using NE, the mechanical length constant, {lambda}, was estimated by fitting an exponential model to the data:

Formula (1)
where {Delta}D is the reduction in the internal diameter at the distance x from the stimulation site, and b is a constant. Parameter estimation was performed using the nonlinear function module in Statistica® (vers. 5.1, Statsoft, OK). Mechanical length constants were estimated for each individual experiment under baseline conditions, and then averaged to give a mean mechanical length constant for all arterioles. Pearson’s correlation coefficient was calculated using the standard formula [12].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Stimulation with NE
Mean arterial blood pressures prior to infusion of vasoactive substances (101–106 mmHg) did not differ significantly between the ANG II-, methoxamine-, and losartan groups, respectively. Mean blood pressure increased by 10.4±1.6 mm Hg during infusion of methoxamine (P<0.05), and by 6.2±1.6 mm Hg during infusion of ANG II (P<0.05). The difference in blood pressure responses to infusion of vasoconstrictors was not statistically significant. Blockade of endogenous ANG II by losartan decreased mean blood pressure by 6.6±1.3 mm Hg (P<0.05).

Mean resting internal diameters of the arterioles at the stimulation site before and after infusion of vasoactive agents are shown in Table 1. The values in the different groups were not statistically significant. No significant changes in the mean resting arteriolar diameters were induced by systemic infusion of either ANG II, methoxamine or losartan. Also, the local reduction in internal diameter after application of NE was similar in the three groups both before and after infusion of either ANG II, methoxamine or losartan (Table 1). As shown in Table 1, the application rate of NE necessary to achieve an approximately 50% reduction in inner diameter was similar in the three groups, and it did not change significantly following the infusion of the vasoactive substances.


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  		Table 1 Arteriolar local resting internal diameters, local reaction to microapplication of NE and electrical stimulation, and rate of NE and voltage delivered from the pipettea

 
Fig. 2 shows typical time courses for the local response and the response at 600 µm upstream from the application site. The two responses developed in parallel, and a stable response was achieved within approximately 20 to 25 s after starting the application of NE. In a few experiments, the local responses were initially unstable requiring up to 1.5 min of NE application before the local response had stabilized.


Figure 2
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  		Fig. 2 Time course for development of local and conducted (600 µm upstream) vasoconstriction to micropipette delivery of NE. Arrow indicates start of NE application. These data were obtained during two separate applications of NE in the same vessel. Upper panel: Diameter (D) given in absolute numbers. Lower panel: Diameters normalized to the resting diameter at 0 and 600 µm, respectively.

 
Local and conducted vasoconstrictor responses to NE before and after infusion of ANG II, methoxamine, and losartan are shown in Fig. 3 A–C. There were no significant differences between the baseline (i.e., before infusion of vasoactive substances) conducted vasoconstrictor responses in the three groups (ANOVA). When the baseline conducted responses from the three groups were pooled, the mean mechanical length constant, obtained from non-linear estimation in individual experiments, was 998±130 µm (n=31). Since responses measured close to the stimulation site (<350 µm) may result from a combination of conducted vasoconstriction and diffusion of the stimulating agent it has been suggested that these measurements should be excluded from analyzes of mechanical length constants [5]. However, using only the conducted responses at 400–1000 µm to calculate length constants did not significantly change the result. The conducted vasoconstrictor response to NE was significantly increased by infusion of ANG II (P<0.02, ANOVA, Fig. 3A). Infusion of methoxamine did not alter conducted vasoconstrictor responses to NE at any of the measured sites (Fig. 3B). In contrast, blockade of the effects of endogenous ANG II by administration of the AT1-receptor antagonist losartan decreased conducted responses to NE almost to the extent where remote responses were undetectable (P<0.03, ANOVA, Fig. 3C).


Figure 3
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  		Fig. 3 Local and conducted responses after application of NE. Change in arteriolar internal diameter ({Delta}D) induced by application of NE as a function of the distance from the stimulation site before (dashed lines) and after (solid lines) intravenous infusion of: (A) ANG II (n=13 vessels), (B) methoxamine (n=10 vessels), and (C) losartan (n=8 vessels). Vertical bars indicate±SE. * P<0.05.

 
3.2 Local electrical stimulation
Infusion of ANG II increased mean arterial blood pressure from 109±3 to 119±4 mmHg (n=6, P<0.05) and infusion of losartan decreased blood pressure from 102±3 to 92±4 mmHg (n=6, P<0.05). Mean arteriolar resting internal diameters are summarized in Table 1. The local reduction in internal diameters and the applied voltages did not differ between groups, and both remained unchanged following administration of either ANG II or losartan (Table 1).

Conducted vasoconstrictor responses 600 µm upstream from the pipette before and after infusion of ANG II and losartan are shown in Fig. 4. ANG II significantly increased the conducted response from 7.7±0.9 µm to 9.3±0.9 µm (P<0.05). Blockade of endogenous ANG II by losartan decreased conducted vasoconstriction from 6.6±0.7 µm to 2.8±0.8 µm (P<0.001).


Figure 4
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  		Fig. 4 Conducted responses after local electrical stimulation. Change in arteriolar internal diameter induced by local electrical stimulation measured 600 µm upstream from the stimulation site before and after ANG II (n=6) and losartan (n=6). Vertical bars indicate SE. * P<0.05; ** P<0.01.

 
3.3 Effect of change in the arterial pressure
The absolute difference in vasoconstriction measured 600 µm upstream before and after administration of ANG II or losartan (data for NE and electrical stimulation pooled) were not correlated to the absolute change in arterial blood pressure induced by infusion of either ANG II or losartan (Fig. 5, n=33, r2=0.12, NS).


Figure 5
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  		Fig. 5 Absolute differences, (|{Delta}Dc–{Delta}De|), between conducted vasoconstrictor responses 600 µm upstream from the pipette before ({Delta}Dc) and after ({Delta}De) infusion of ANG II or losartan versus the corresponding absolute changes in mean arterial pressure (MAP) ({Delta}MAPc–{Delta}MAPe); (n=33), r2=0.12, NS. {diamondsuit} Stimulation: NE, infusion: ANG II; {blacksquare} stimulation: NE, infusion: losartan; {blacktriangleup} stimulation: current, infusion: ANG II; x stimulation: current, infusion: losartan.

 
3.4 Influence of veins on conducted vasoconstriction
In most cases (20 out of 31 vessels) the arterioles stimulated by NE had no accompanying vein or venule (see Fig. 1). In the present study, an accompanying vein/venule was defined as any vein/venule within 200 µm of the arteriolar segment under investigation. The conducted vasoconstrictor responses did not differ between vessels with and without an accompanying vein/venule (Table 2).


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  		Table 2 Local and conducted responses (relative reduction in diameter) during the control period in all NE experiments grouped according to whether the stimulated arteriole had an accompanying vein or nota

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusion
 References
 
The main finding of the present study is that conducted vasoconstriction to either locally applied norepinephrine or electrical current is increased after systemic infusion of ANG II and reduced after blockade of endogenous ANG II by administration of the AT1-receptor antagonist losartan. In contrast, infusion of the {alpha}1-agonist methoxamine had no effect on conducted vasoconstriction.

4.1 Mechanisms of remote vasoconstriction
The remote vasoconstrictor responses observed in the present study could not be explained by either diffusion or convection of NE to the upstream sites. Slight retraction of the pipette abolished both the local and the remote responses, and even prolonged application of NE at high rates (20 nl/min) did not induce a local or remote response. This conclusion is also supported by the time course of the development of vasoconstriction (Fig. 2). Both the local and the remote responses appear almost instantaneously after initiating the NE application, and both reach a steady state after approximately 20–25 s. The observed time courses are in good agreement with those of previous studies of conducted vasodilation using sustained stimulation [14,15].

Some studies have shown that remote responses in arterioles can be caused by upstream convection of agonists by adjacent veins or venules [7,16]. However, this mechanism does not play a significant role in the present experiments, since the remote responses were similar in arterioles with and without an accompanying vein/venule (Table 2). It therefore appears that the remote responses observed in the present study are caused by so called conducted vasoconstriction. It is currently believed that this results from electrotonic propagation of the change in membrane potential induced at the site of stimulation, where the subsequent constriction at the remote site are due to electromechanical coupling [17,18]. Vasoconstriction was induced by application of two different stimuli, NE and electrical current. NE causes vasoconstriction through activation of {alpha}1-adrenergic receptors on the smooth muscle cells. This may be mediated through both electromechanical and pharmacomechanical coupling, where the latter signifies contraction of smooth muscle cells independent of changes in membrane potential. Conducted vasoconstriction, however, is likely to rely on electromechanical coupling alone [18].

Electrical stimulation directly induces a local depolarization of the smooth muscle and endothelial cells. This would give rise to conducted vasoconstriction through an electrotonic spread along the smooth muscle and/or the endothelial cells. Theoretically, both the local and the conducted responses to electrical stimulation could also be caused, at least in part, by release of vasoactive substances such as NE from perivascular nerves. However, in a recent study Steinhausen and coworkers found that conducted responses to local electrical stimulation were unaltered after addition of either the {alpha}1-adrenergic receptor antagonist prazosin, or tetrodotoxine (TTX), a blocker of neural fast sodium channels [19]. This study was performed in rat renal afferent arterioles which like the mesenteric arterioles has a rich supply of perivascular sympathetic nerve fibers [20]. Thus, it seems unlikely that release of vasoactive neurotransmitters from perivascular nerves played a significant role when vasoconstriction was induced by electrical stimulation via micropipettes.

4.2 Effect of changes in the arterial blood pressure
In the present study we perturbed the renin–angiotensin system by either infusion of ANG II so as to increase the plasma levels of ANG II, or by injection of the AT1-receptor antagonist losartan. As expected, increasing the plasma levels of ANG II increased the mean arterial pressure, whereas inhibition of the effects of endogenous ANG II through AT1-receptor blockade reduced blood pressure. Although the changes in mean arterial pressure were modest (≤10 mm Hg), it could be argued that the observed effects on conducted vasoconstriction were secondary to changes in arterial blood pressure and/or vascular tone [21]. To test this possibility a second vasopressor agent, methoxamine, was also infused. Infusion of methoxamine increased arterial blood pressure slightly more than ANG II, but had no effect on the conducted vasoconstriction. Methoxamine is an {alpha}1-adrenergic agonist with no β-adrenergic stimulating effects, and it seems reasonable to assume that the increase in vascular tone exceeded that in the ANG II-treated animals. The local increase in vasomotor tone could, however, still be greater after infusion of ANG II, since ANG II and methoxamine have different efficacies at different levels of the microcirculation [22]. To further assess the role of the changes in arterial pressure we compared the effect on the conducted response to the change in the mean arterial pressure after either ANG II or losartan in the individual experiments. As is evident from Fig. 5 there was no correlation between the absolute change in the blood pressure and the absolute change in the conducted response at 600 µm. This was the case regardless of whether NE or an electrical current was used as stimulus.

Finally, it should be considered that the infusions of neither ANG II nor losartan caused measurable changes in the resting diameters of the experimental vessels (cf. Table 1). This is to be expected when one considers the modest increase in total peripheral resistance needed to explain the observed increases in arterial pressure together with the inverse proportionality between the fourth power of the vascular radius and the resistance (Pouisseuile’s law). Thus, it seems unlikely that the observed effects of ANG II and losartan on conducted vasoconstriction can be explained simply by changes in the arterial pressure and/or the arteriolar tone.

4.3 Potential mechanisms for the modulating effect of ANG II
The mechanism(s) by which ANG II augments conducted vasoconstrictor responses in mesenteric arterioles are not known. There appears to be several possibilities. One mechanism could be through sensitization of the contractile apparatus of the smooth muscle cells to the propagated stimulus [23]. If ANG II specifically increased the electromechanical coupling of the vascular smooth muscle cells this could explain the increased conducted vasoconstriction observed in our experiments. However, this seems unlikely for the following reason: If ANG II had increased the sensitivity of the smooth muscle cell contractile apparatus to membrane depolarization (the electromechanical coupling) an increased local response to electrical stimulation after ANG II would have been expected. However, the local responses to both NE and electrical stimulation were unchanged after infusion of ANG II suggesting that ANG II in the dose used did not alter neither pharmaco- nor electromechanical coupling.

Another possibility is that ANG II increases the longitudinal electrical conductance (electrical length constant) of the endothelium and/or the smooth muscle cells of the arteriole. Such an effect would extend the electrotonic spread of the depolarization induced at the pipette to a larger part of the vessel. This possibility is not inconsistent with the fact that the curves in Fig. 3A and C appear to be parallel in the range from 400 to 1000 µm before and after infusion of either ANG II or losartan. The lower of the two curves has a greater fractional decline than the upper curve, and if redrawn in a semilogarithmic plot, the slope of the lower curve exceeds that of the upper curve. This is also evident when comparing the fractional decline in the conducted responses between 400 and 1000 µm. In the ANG II group the response at 1000 µm increased from 47% to 59% of that at 400 µm following ANG II infusion. In contrast, in the losartan group the corresponding response decreased from 45% to 9%.

An increased arteriolar electrical length constant could result either from an increased cell-to-cell coupling through an upregulation of the function or number of endothelial- or smooth muscle cell gap junctions or from an increase in membrane resistance. Further experiments are required to specifically test this hypothesis and to determine whether gap junctional communication or membrane resistance are in fact increased by ANG II in this preparation. However, regardless of the underlying mechanism the modulating effect of ANG II on the conduction process could be of significance in steady-state microcirculatory control.


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
References
 
The present study shows that rat mesenteric arterioles are suitable for studying conducted vasoconstriction. In addition, it is shown that conducted vasoconstriction to norepinephrine and local electrical stimulation are modulated by ANG II. An increase in the plasma levels of ANG II caused an increase in conducted vasoconstrictor responses, whereas blockade of the endogenous ANG II by the AT1-receptor antagonist losartan decreased the conducted response. The {alpha}1-adrenergic agonist methoxamine, yielding comparable blood pressure responses to that of ANG II, had no effect on conducted vasoconstrictor responses.

Time for primary review 23 days.


   Acknowledgements
 
The authors wish to thank Anni Salomonsen, Ian Godfrey, Nina Buch and Eva C. Heins for excellent technical assistance and Dr. Jørn Hounsgaard for technical advice. Losartan was kindly provided by Merck Sharp & Dohme. The study was supported by the Danish Heart Foundation, the Danish Medical Research Council, the Jacob and Olga Madsen Foundation, the John and Birthe Meyer Foundation, and the Novo-Nordisk Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 

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