Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.
Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.
1. Surgical Excision of the Small Intestinal Mesenteric Vascular Arcade
2. Dissection and Cleaning of Arteries
3. Cell Isolation for Patch Clamp Studies
4. Use of Whole Cell Patch Clamp to Measure Kv7 Potassium Currents or Membrane Voltage
5. Use of Intact Artery Segments for Pressure Myography
6. Representative results
The results shown in Figure 3 illustrate typical recordings of Kv7 currents from mesenteric artery myocytes using a voltage step protocol before (Figure 3A,B black traces) and during treatment with a Kv7 channel activator Zn pyrithione (ZnPyr, 100 nM; Figure 3A,B red traces). Similar voltage step data from 3 cells were averaged to prepare the current-voltage (I-V) plots shown in Figure 3D. A representative time course of enhancement of current amplitude (measured by continuous voltage clamp at -20 mV) during treatment with 100 nM Zn pyrithione is shown in Figure 3C. Figure 4B illustrates the time course of the effect of ZnPyr on mesenteric artery outer diameter measured using pressure myography; the vessel was pre-constricted with 100 pM arginine vasopressin (AVP) prior to addition of 100 nM ZnPyr. Averaged dose-response data for ZnPyr-induced mesenteric artery dilation are shown in Figure 4C.
Figure 1. Picture of a mesenteric arcade in the dissecting chamber.
Figure 2. Picture of a myocyte with a patch pipette on its surface.
Figure 3. Original traces of Kv7 currents, time course of drugs application, I-V curves. A. Family of sequential current traces (lower panel) recorded in response to voltage steps (top panel) before treatment (control, black) and following stabilization in the presence of 100 nM ZnPyr (red) in a single MASMC. B. Representative current traces from A (as indicated by arrowheads) for control (black) and for 100 nM ZnPyr (red). Gray bars indicate time interval where averaged current amplitudes were measured for current-voltage (I-V) plots. Step voltages are indicated on the right by arrowheads. C. Time course of Kv7 current enhancement by 100 nM ZnPyr measured with continuous voltage clamp at -20 mV (bar). D. Average I-V curves (n=3) derived from Kv7 current densities recorded before (control, filled circles), during treatment with 100 nM ZnPyr (open circles), and during treatment with 10 μM XE991 (filled triangles). Non-specific leak currents were subtracted as described by Passmore et al. 1.
Figure 4. ZnPyr induced relaxation of a mesenteric artery pre-constricted with 100 pM AVP. A. Photograph of a mesenteric artery mounted in the pressure myograph set-up. B. Time course of changes in outer diameter of the vessel in response to application of 100 pM AVP (open bar) followed by application of the 100 nM ZnPyr (filled bar). C. Bar graph summarizing results of 100 nM ZnPyr-induced vasodilaion.
The methods and experimental approaches described here are quite robust and can produce clear and reproducible results when applied with meticulous attention to detail. Good electrophysiological recordings and constriction/dilation of arterial segments are dependent on the health of the cells and artery segments, respectively. Cell preparations can vary from day to day, even using the same protocol. Isolation Solutions can be used for up to 2 weeks, but if the quality of the cell preparation is low on two consequent days new Isolation Solutions should be prepared. We have found that enzyme activities vary from batch to batch and therefore isolation conditions (the times of incubation and concentrations of enzymes) require optimization with each new batch of enzymes. We have also found that the cell isolation protocol described here for rat mesenteric artery is not optimal for other vascular beds or other species, which may have a different mix or density of extracellular matrix components. Each vascular preparation requires its own optimization to identify conditions that produce the healthiest cells. The criteria that we find most helpful in identifying healthy cells are: 1) cell morphology: smooth, elongated, but slightly constricted appearance after triturating MA segments in the Isolation Solution containing 50 μM Ca2+, and retaining the same appearance when transferred to 2 mM Ca2+-containing external solution. Not all cells will retain their nearly fully relaxed elongated shape, but only nearly fully relaxed myocytes should be used for electrophysiological recording; 2) Cells should be optically dense, which indicates an intact undamaged membrane; 3) Resting membrane voltages of healthy myocytes used to record potassium currents or for current-clamp recordings of membrane voltage should be more negative than -40 mV (usually in the range of -45 mV to -56 mV under the ionic conditions described in our protocol here).
To ensure successful electrophysiological recordings, the bath solution and the pipette solution (for composition see Table 1) should be prepared on the day of the experiment. It is crucial to adjust osmolality of the both internal and bath solutions to be identical (298-299 mOsM).
Isobaric arterial function measurements made in small arteries (<500 μm) using pressure myography more closely approximate physiological conditions than do isometric force measurements made using a wire myograph. The vessels in a pressure myograph are typically more sensitive to vasoconstrictor agonists2-4, more closely approximating the sensitivities measured in vivo5, 6. The increased sensitivity to low concentrations of agonists has enabled the identification of signal transduction mechanisms induced by physiologically relevant picomolar concentrations of AVP7, 8.
Pressure myography preserves the geometry of the vessel and maintains the integrity of the endothelium. Endothelium-mediated effects of drugs can be distinguished from smooth muscle-mediated effects by conducting parallel measurements in intact and endothelium-denuded vessels9. The intentional disruption of the endothelium can be accomplished by physically damaging the endothelium (e.g. by passing a human hair back and forth through the lumen) or by perfusing air through the lumen of the artery. The loss of endothelial function (but not vascular smooth muscle function) should be confirmed by measuring a reduced endothelium-mediated vasodilator response to acetylcholine in vessels pre-constricted with an agonist9.
Parallel measurements of ionic currents/membrane voltage in mesenteric artery myocytes using patch clamp techniques and arterial constriction/dilation of mesenteric arteries using pressure myography can enable elucidation of the roles of specific ion channels in both physiological signal transduction cascades and in the actions of therapeutic agents. Treatments targeting specific classes of ion channels or specific signal transduction intermediates can be applied additively or in sequence to provide mechanistic information about the roles of these channels in the regulation of myocyte function at the cellular level and in constriction/dilation of intact arteries. Convergent results of treatments that enhance or inhibit particular types of ionic currents with corresponding effects on artery diameter provide more reliable interpretations than can be obtained using either method by itself. Ideally, the concentration-dependence of the agents under study should be determined in both systems. To assess physiological or therapeutic relevance, the concentrations tested should be related to concentrations measured in the physiological environment or achieved with clinical therapies. Examples of these types of experimental approaches can be found in our previous publications8-10.
The authors have nothing to disclose.
This work was funded by a grant from National Heart, Lung, and Blood Institute (NIH R01-HL089564) to KLB and pre-doctoral fellowships from the American Heart Association (09PRE2260209) and Arthur J. Schmitt Foundation to BKM.
Name | Company | Catalog Number | Comments |
Sodium Chloride | Sigma | S5886 | Dissecting Solution: 145 Bath solution for Electrophysiology*: 140 Internal solution for electrophysiology: 10 Isolation solution for myocytes*: 140 Bath solution for pressure myography: 145 Lumen solution for pressure myography: 145 |
Potassium chloride | Sigma | P5405 | Dissecting Solution: 4.7 Bath solution for Electrophysiology*: 5.36 Internal solution for electrophysiology: 135 Isolation solution for myocytes*: 5.36 Bath solution for pressure myography: 4.7 Lumen solution for pressure myography: 4.7 |
Potassium EGTA | Sigma | E4378 | Internal solution for electrophysiology: 0.05 |
HEPES | Sigma | H9136 | Bath solution for Electrophysiology*: 10 Internal solution for electrophysiology: 10 Isolation solution for myocytes*: 10 |
Disodium hydrogen phosphate | Sigma | S5136 | Isolation solution for myocytes*: 0.34 |
Potassium hydrogen phosphate | Sigma | P5655 | Isolation solution for myocytes*: 0.44 |
Magnesium Chloride | Sigma | M2393 | Bath solution for Electrophysiology*: 1.2 Internal solution for electrophysiology: 1 Isolation solution for myocytes*: 1.2 |
Calcium Chloride | Sigma | C7902 | Bath solution for Electrophysiology*: 2 Isolation solution for myocytes*: 0.05 |
Sodium phosphate | Fisher Scientific | BP331-1 | Dissecting Solution: 1.2 Bath solution for pressure myography: 1.2 Lumen solution for pressure myography: 1.2 |
Magnesium Sulfate | Sigma | M2643 | Dissecting Solution: 1.17 Bath solution for pressure myography: 1.17 Lumen solution for pressure myography: 1.17 |
MOPS | Fisher Scientific | BP308 | Dissecting Solution: 3 Bath solution for pressure myography: 3 Lumen solution for pressure myography: 3 |
Pyruvic acid | Sigma | P4562 | Dissecting Solution: 2 Bath solution for pressure myography: 2 Lumen solution for pressure myography: 2 |
EDTA dihydrate | Research Organics | 9572E | Dissecting Solution: 0.02 Bath solution for pressure myography: 0.02 Lumen solution for pressure myography: 0.02 |
D-Glucose | Sigma | G7021 | Dissecting Solution: 5 Bath solution for Electrophysiology*: 10 Internal solution for electrophysiology: 20 Isolation solution for myocytes*: 10 Bath solution for pressure myography: 5 Lumen solution for pressure myography: 5 |
Bovine serum albumin | Sigma | A3912 | Dissecting Solution: 1% Lumen solution for pressure myography: 1% |
pH | Dissecting Solution: 7.4 Bath solution for Electrophysiology*: 7.3 Internal solution for electrophysiology: 7.2 Isolation solution for myocytes*: 7.2 Bath solution for pressure myography: 7.4 Lumen solution for pressure myography: 7.4 |
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Osmolarity | Dissecting Solution: 300 Bath solution for Electrophysiology*: 298 Internal solution for electrophysiology: 298 Isolation solution for myocytes*: 298 Bath solution for pressure myography: 300 Lumen solution for pressure myography: 300 |
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*11 |
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Table 1. Components of solutions used in the experiment. |