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Eur J Cardiothorac Surg 2008;34:109-112. doi:10.1016/j.ejcts.2008.03.053
Copyright © 2008, European Association for Cardio-thoracic Surgery. Published by Elsevier. All rights reserved.
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Transmural pressure and membrane potential in human saphenous vein

Detlef Biegera, Carol Ann Forda, Kam Mongb, Reza Tabrizchia,*

a Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, NL, Canada
b Discipline of Surgery, Health Care Corporation of St. John's, St. John's, NL, Canada

Received 24 September 2007; received in revised form 26 March 2008; accepted 27 March 2008.

* Corresponding author. Address: Division of BioMedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, Health Sciences Centre, St. John's, NL, Canada A1B 3V6. Tel.: +1 709 777 6864; fax: +1 709 777 7010. (Email: rtabrizc{at}mun.ca).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: An increase in transmural pressure reportedly depolarizes myocytes in various arterial blood vessels. We have examined the relationship between transmural pressure and membrane potential (E m) in human saphenous veins with a view to determine whether contractile force generation, hence spasmogenesis in vein grafts, involves a similar process of mechanoelectrical excitation. Methods: Intracellular recordings were made by sharp glass microelectrodes in human isolated saphenous veins and parallel measurements were performed in ring preparations. Results: E m values obtained in pressurized vessels at four different pressure levels were (mean ± SD): –74.4 ± 5.5 mV (0–6 cm H2O; n = 10), –72.6 ± 6.5 mV (11–14 cm H2O; n = 27), –72.1 ± 6.5 mV (26–27 cm H2O; n = 30), and –72.9 ± 4.0 mV (50–54 cm H2O; n = 38), demonstrating the lack of an overt pressure-dependence. Except at the lowest transmural pressure tested, these values were significantly different from E m obtained in ring preparations (–77.8 ± 4.0 mV; n = 30). Raising extracellular K+ to 80 mM produced a comparable depolarization in tissues either pressurized to 50–54 cm H2O (–64.9 ± 4.3 mV; n = 27) or set up as ring preparations (–64.06 ± 6.9 mV; n = 35). Conclusions: Human saphenous veins respond to transmural pressure with a limited depolarization that lacks correlation with pressure. The absence of a pressure-induced graded depolarization suggests that pressure-dependent vasoconstriction does not play a primary role in blood flow regulation in lower limb large veins. Moreover, this raises doubts that mechanical stimuli per se would lead to development of vasospasm in the early stages of saphenous vein grafting into arterial vascular beds.

Key Words: Vascular smooth muscle • Pressurized vessels • Ring preparation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The saphenous vein is the most commonly used conduit in coronary artery bypass surgery but its occlusion rate within 6 months can be greater than 20% [1]. Possible causes of occlusion include thrombus formation due to endothelial cell damage and spasm [2], the latter conceivably ensuing from pressure-dependent vasoconstriction following grafting into the arterial circulation. Spasmogenesis may involve different mechanisms, such as mechanoelectrical and/or pharmacomechanical coupling. In either case, depolarization of vascular myocytes would lead to the opening of voltage-gated L-type calcium channels and calcium influx, resulting in vasoconstriction and, potentially, graft occlusion. L-type calcium channels of human saphenous veins have been characterized as having a low threshold of activation [3]. Hypothetically, if pressure-dependent vasoconstriction existed in human saphenous veins, this would imply the presence of ion channels that induce depolarization of membrane potential (E m) in response to mechanical stimulation.

In arterial beds, pressure-induced depolarization is recognized as a physiological phenomenon that is responsible for vasoconstriction [4] and ultimately autoregulation of blood flow. In pressurized vessels, an increase in transmural pressure has been reported to result in depolarization of smooth muscle cells. For instance, Smeda and Daniel [5] have reported a significant depolarization of smooth muscle cells following an increase in transmural pressure in arterial segments from canine ileum with E m shifting from –54.7 ± 2.2 mV at 40 mmHg to –39.0 ± 1.7 mV at 160 mmHg. Similar findings have also been described in mouse cerebral [6] and rat mesenteric arteries [7].

While myocyte E m in arterial high-pressure vessels is sensitive to an increase in transmural pressure, the same may not apply to low-pressure vasculature such as limb veins. Though widely employed as a graft, especially in coronary artery bypass procedures [8], the human saphenous vein is less well known in terms of its electrophysiological properties. Here, we have examined the effect of different transmural pressures on E m of human isolated saphenous vein as determined by sharp glass microelectrode recording [9]. For comparison, E m was also recorded in conventional ring preparations. In addition, we assessed the impact of high extracellular potassium ([K+]e) on changes in E m in pressurized vessels as opposed to ring preparations.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
Experiments conducted for this investigation conformed to Canadian Institutes of Health Research Guidelines on Research Involving Human Subjects. The protocol for these experiments was approved by the Human Investigation Committee, Memorial University of Newfoundland.

2.1 Tissue isolation
Experiments on surplus saphenous veins for all the studies described were performed on the same day on which they were taken from patients. Segments of saphenous veins were obtained from a total of 31 patients (20 males and 11 females; age range 49–78 years) undergoing coronary artery bypass surgery. The tissues were placed in ice-cold physiological salt solution immediately after leaving the operating room. The composition of the physiological salt solution was the following (in mM): NaCl, 132; KCl, 4.0; MgCl2, 1.2; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 12.5; ethylenediaminetetraacetic acid, 0.1; glucose, 11.0. The veins were cleaned of connective and adipose tissue in ice-cold physiological salt solution. For pressurizing vessels polyethylene tubing (I.D. 0.58 mm; O.D. 0.965 mm) was inserted into the lumen of both sides of the saphenous vein of 1–2 cm in length with one tube connected to a pressure transducer and the other connected to a reservoir containing physiological buffer the height of which could be changed to a desired pressure. Ring preparations were cut into 2 mm lengths mounted unstretched in an inside-out configuration by means of stainless steel minutien pins in a temperature-controlled perfusion chamber.

2.1.1 Membrane potential measurements
Saphenous vein blood vessels (pressurized or rings) were held in place in a 5 ml Sylgard-lined tissue chamber perfused with 95% O2: 5% CO2 pregassed physiological salt solution of pH 7.4, delivered under a constant pressure head at a rate of 3–4 ml per min and warmed to 35 ± 1 °C. Blood vessels were allowed to equilibrate for 60 min. E m was recorded with borosilicate thick-walled capillary microelectrodes that were pulled on a Flaming Brown micropipette puller (Model P-80-PC, Sutter Instrument Co. CA, USA) and filled with 3 M KCl to yield a tip resistance of 10–20 M{Omega}. An Ag/AgCl reference half-cell containing KCl (3.0 M) was connected to the bath via an agar salt bridge containing 150 mM NaCl. Impalements were made by means of a Narishige x–y–z micropositioner at depths >50 µm below the surface, from the adventitial side for pressurized preparations and typically from the endothelial side and at times from the cut edge of the ring-mounted vessel. Usually 4–12 different cells were sampled at various pressure levels in regular [K+]e buffer and after switch-over from low to high [K+]e. Criteria for successful impalement and measurement of smooth muscle cell E m were an abrupt drop in voltage upon penetration of the cell membrane, a stable potential for at least 3–4 min, and a sharp return to zero upon withdrawal of the electrode. Voltage signals were recorded by means of an Axoclamp-2A (Axon Instruments Inc. Burlington, CA, USA), the output of which was fed into a DigiData 1200 Series Interface (Axon Instruments Inc.). Electrophysiological studies were only carried out if tissues were mechanically responsive to an elevation in [K+]e to 80 mM.

The transmural pressure in the saphenous vein was changed by elevating or lowering the reservoir to 0–6, 11–14, 26–27 and 50–54 cm H2O (1.359 cm H2O = 1.0 mmHg). The sequence of changes in transmural pressure was random. The changes in pressure were monitored using a pressure transducer (Gould Statham, USA; Model P23B) connected to a custom built amplifier. Both electrophysiological and pressure data were acquired and displayed on AxoScope (Version 1.1) and stored on a computer hard disk.

The E m was also recorded after raising K+ concentration (80 mM; with Na+ being replaced iso-osmotically as the concentration of K+ increased), i.e. within 5–10 min and thereafter for up to 1/2–1 h) in pressurized (50–54 cm H2O) blood vessels and ring preparations. The pH was 7.4 when the solution was saturated with a 95% O2–5% CO2 gas mixture at 35 ± 0.5 °C. Cells were sampled after changes in buffers and each group of experiments included five to six different blood vessels from different patients.

2.2 Statistical analysis
The experimental design and analyses were random, unbiased, and no group assignments were done according to patient sex, disease state or drug history. For each experimental group, electrophysiological data were obtained from 4 to 12 cells in each surgical specimen (one per patient). One-way analysis of variance was employed for comparisons between means, and the Student–Newman–Keuls test was used for pairwise comparisons to identify differences between individual means. In addition, linear regression analysis followed by analysis of variance of the effect of changes in pressure on E m was carried out. For all cases, a probability of error of less than 0.05 was selected as the criterion for statistical significance. Unless noted otherwise, data are presented as the mean ± SD.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
E m values for vascular smooth muscle cells of human saphenous vein are presented in Fig. 1 . When compared to values obtained in pressurized blood vessels, the E m recorded in ring preparations showed an apparent small hyperpolarization, which was evident at all pressure levels tested, except for the lowest. While no significant differences were found between E m recorded in ring preparations and pressurized vessels at 0–6 cm H2O (p = 0.134), regression analysis of the effect of changes in pressure on E m indicates an R of 0.000260, R 2 of 0.0000000818 with adjusted R 2 being zero. Thus, there were no significant differences found between E m values recorded at different pressures.


Figure 1
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  		Fig. 1. Membrane potential of smooth muscle cells of human saphenous veins recorded in ring preparation (cells; n = 30), and vessels pressurized at 0–6 cm H2O (n = 10), 11–14 cm H2O (n = 27), 26–27 cm H2O (n = 30) and 50–54 cm H2O (n = 38) from 3 to 6 different tissues. Each value is the mean ± SD. aSignificantly different from ring preparation; p < 0.05.

 
Raising [K+]e to 80 mM resulted in a modest depolarization of smooth muscle cells in ring preparations, which was of a magnitude similar to that previously reported [3]. Moreover, a corresponding depolarization was observed in high K+ (80 mM) solution in tissues pressurized to 50–54 cm H2O (Table 1 ). Thus, E m values of cells in ring preparations and pressurized vessels in high K+ solution did not differ significantly.


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  		Table 1 Membrane potential (E m) of smooth muscle cells of human saphenous veins in ring preparation or pressurized (50–54 cm H2O) vessels in normal buffer and high K+ (80 mM) solution
 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The present study demonstrates the absence of a pressure-dependent depolarization in human saphenous veins. An increase in the transmural pressure (0–54 cm H2O) as employed in this investigation did not result in a graded depolarization of the smooth muscle cells. As argued in the introduction, pressure-dependent alteration in E m with secondary activation of voltage-gated calcium channels and calcium influx might contribute to the development of vasospasm and ultimately loss of patency in saphenous vein grafts. In light of our data, it seems unlikely that mechanical stimulation (mechanoelectrical excitation) is the main cause for the development of vasospasm in the early stages after saphenous vein grafting. It is pertinent to note that voltage-gated calcium channels in this tissue are likely to belong to a type characterized by a low threshold of activation [3]. Accordingly small decreases in E m may result in the activation of contractile force generation. Such effects could be mediated by chemical signals instead of physical stimuli (see below).

The resting E m value of smooth muscle of saphenous veins as recorded from isolated ring preparations has been previously reported as –62 ± 0.9 mV [10,11]. This value is considerably more depolarized than the present and previously reported data from our laboratory [3,12]. There is a possibility of a sampling bias in the present investigation as smooth muscle cells preferentially sampled in ring preparations may have been in closer proximity to the endothelial cell layer. Specifically, a more hyperpolarized resting E m recorded from the smooth muscle cells in the ring preparations may have been due to the impact of factors released from the endothelial cells. The previously reported resting E m of –77 ± 0.7 mV for smooth muscle cells of saphenous veins in ring preparations under a tension of 19.6 mN [12] is not significantly different from values obtained in ring preparations in this investigation. Interestingly, the resting E m of dissociated smooth muscle cells from human saphenous vein is reported as –41 ± 2.0 mV [13]. Thus, the E m recorded from dissociated smooth muscle cells is much less negative when compared to recordings from the isolated blood vessel segments, suggesting that dissociation of cells from their matrix produces extensive depolarization, an issue that could be important when studying the physiology and pharmacology of this type of cells.

On the basis of Nernst equation, the equilibrium potential for K+ should have a slope of approximately 58 mV per log unit of an increase in [K+]e [14,15]. This is evidently not the case in human saphenous vein. We previously reported that the change in E m induced by increasing [K+]e amounts to approximately 15 mV per 10-fold change in [K+]e [3]. Furthermore, such a discrepancy has also been reported for other blood vessels, for example, portal vein [16,17], canine carotid artery [18], guinea-pig superior mesenteric artery [19], and coronary artery [20]. However, as previously reported, a very steep electromechanical relationship exists for human saphenous veins where force generated in buffer containing high K+ revealed a 3.62 mN force per mV of change in E m [3]. This apparently limited depolarization of vascular smooth muscle cells to high [K+]e has been suggested to be due to low conductance for K+ [21]. In light of the present study, it also seems clear that an increase in transmural pressure does not readily translate into an increase in K+ conductance and, hence, to an increase in the slope relating to E m and [K+]e. Clearly, changes in transmural pressure did produce modest yet significant depolarization in comparison to the unstretched preparation, however, this small change failed to alter responsiveness to high [K+]e.

In the present investigation, the highest transmural pressure to which the veins were exposed corresponded to ~38 mmHg, equivalent to the gravitational pressure that veins experience in vivo at the upper leg level in man in a standing position. Venous pressure in the human saphenous vein is reported to range from 6.7 to 8.8 mmHg in a horizontal position [22,23], while tilting can result in an increase in venous pressure in the order of 47.0 mmHg [22].

Modest but significant depolarization of E m, as induced by an increase in transmural pressure in the vessels, may facilitate the response to neurotransmitters and humoral factors. However, based on the present data in human lower limb veins there does not appear to be a significant shift in the E m as transmural pressure increased from 10 to 38 mmHg. Evidently, these data contrast with findings in mammalian arteries where transmural pressure results in a sizeable depolarization of vascular smooth muscle cells. For example, in cat isolated cerebral arteries a change from 0 to 150 mmHg in transmural pressure resulted in a drop of the resting E m from –68 ± 1.6 mV (mean ± SEM) to significantly less negative values of ~–30 mV [24]. Moreover, the steepness of pressure-induced depolarization varied with the concentration of extracellular Ca2+ [24], and was greatly diminished by removal of the endothelium in the cat cerebral arteries [25].

In view of the very limited and discontinuous changes in E m induced by increasing transmural pressure in the human saphenous vein smooth muscle cells, it is unlikely that pressure-dependent vasoconstriction is a primary contributor to blood flow in this segment of the circulatory system. The same argument would hold for this vessel once grafted. The remaining alternative mechanism for regulating contractile force would be pharmacomechanical coupling involving neuro-humoral factors and/or autacoids. Therefore, circulating or neuronally released monoamines might have a significant role in regulating vascular diameter and, hence, blood flow. Further study is needed to determine if modest changes in E m as noted here could act as a trigger to amplify adrenergically mediated responses and significantly affect blood flow in the low-pressure limb of the circulatory system.


   Footnotes
 
{star} This work was supported by a grant-in-aid from Heart and Stroke Foundation of New Brunswick.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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