MATERIALS AND METHODS (supplement for online version)
Cell Isolation: Rabbits and rats were killed with sodium nebutal according to standards of the Canadian Council for Animal Care and the Animal Care Committee of the University of Calgary. Myocytes were isolated as previously described1 and placed in a chamber containing bath solution at room temperature (20-22 °C).
Patch Clamp: C-A and I-O patch clamp methods were employed:2 for the C-A patches, Sylgard-coated pipettes contained (in mmole/L): 140 KCl, 1 CaCl2, 1 MgCl2, 5.5 glucose and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) (pH 7.4 with NaOH; iberiotoxin (IBTX; 200 nmole/L) was added to block large conductance Ca2+-activated K+ channels) and the bath solution contained (in mmole/L): 140 KCl, 2.3 MgCl2, 10 glucose, 1 ethyleneglycol-bis (b-aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA) and 10 HEPES (pH 7.4 with KOH). For I-O patch experiments, the solutions were identical except the bath contained MgATP (0, 0.1 or 1 mmole/L) and MgADP (0.5 mmole/L).
Recordings were performed using an Axopatch 200A amplifier (Axon Instruments, Palo Alto, CA, USA) and pClamp or Axotape software (Axon Instruments). Data were filtered at 2 kHz by an on-board 8-pole Bessel filter before digitization (10-15 kHz). With the exception of panel B of figure 1, all data are displayed with transitions in the outward direction at -40 or -50 mV. Open probability was determined from amplitude histograms (bin width 0.1 to 0.5 pA) based on identical duration recording periods in different treatment groups of 1-3 min and 10-40 s for C-A and I-O patches, respectively. The number of channels in each patches was unknown so open probability (PO) was expressed as NPO (number of channels (N) X mean PO of the single channels) determined according to the following equation:
NPO = (A1 + 2A2 + 3A3 +....+ nAn)/(A0 + A1 + A2 + A3 +....+ An)
where A0, A1, A2, A3 and An are the areas under each histogram peak with the channels closed, one open, and simultaneous openings of 2 to n channels, respectively. Analyses of open dwell time, burst duration and inter-burst interval were made using pClamp software (Axon Instruments). A burst was defined as a train of openings of more than 5 transitions in duration. For statistical analysis, more than 200 individual transitions within at least 5 bursts, and greater than 100 individual bursts from 3 to 4 patches (containing only one channel) were considered for each condition in the determination of mean open and closed times, burst duration and inter-burst interval.
Drugs and Chemicals: PdBu, PdDe, chelerythrine, DNP, 2-DG and glibenclamide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Pinacidil, calphostin C and IBTX were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). Phorbol esters, metabolic inhibitors and PKC inhibitors were added directly to the bath solution. Pinacidil and glibenclamide were prepared in dimethylsulfoxide. Constitutively active PKC (PKM) was prepared by trypsin digestion of purified rat brain PKC as previously described.3 PKC(19-31) was synthesized in the Peptide Synthesis Core Facility of the University of Calgary and purified as previously described.4
Statistics: Paired Student's t test was used for single comparisons and ANOVA followed by Bonferoni’s post-hoc test was used for multiple comparisons. A level of P < 0.05 was considered to indicate a statistically significant difference.
References:
1. Aiello EA, Walsh MP, Cole WC. Phosphorylation by protein kinase A enhances delayed rectifier K+ current in rabbit vascular smooth muscle cells. Am J Physiol. 1995;268: H926-H934.
2. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FS. Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 1981;391: 85-100.
3. Parente, JE, Walsh MP, Kerrick WGL, Hoar PE. Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres. J Muscle Res Cell Motil. 1992;13: 90-99.
4. Light PE, Allen BG, Walsh MP, French RJ. Regulation of adenosine triphosphate-sensitive potassium channels from rabbit ventricular myocytes by protein kinase C and type 2A protein phosphatase. Biochem. 1995;34: 7252-7257.
Table 1 Online: Effect of PKC activation on open probability of Rabbit PV KNDP channels.
Experiment / n / Treatment / NPO1. Pinacidil/Glibenclamide / 3 / Control / 0.005 ± 0.002
Pinacidil (3 mmole/L) / 0.441 ± 0.87
Glibenclamide (3 mmole/L) + Pinacidil / 0.003 ± 001 *
2. Pinacidil/PdBu / 7 / Control / 0.003 ± 0.001
Pinacidil (50 mmole/L) / 0.735 ± 0.178
PdBu ( 50 nmole/L) + Pinacidil / 0.139 ± 0.063 *
3. Pinacidil/Angiotensin II / 4 / Control / 0.0167 ± 0.041
Pinacidil (50 mmole/L) / 0.598 ± 0.069
Angiotensin II (0.1 mmole/L) + Pinacidil / 0.043 ± 0.017 *
4. Pinacidil/PdDe / 5 / Control / 0.002 ± 0.002
Pinacidil (50 mmole/L) / 0.691 ± 0.283
PdDe (50 nmole/L) + Pinacidil / 0.878 ± 0.392
5. Calphostin C Pre-treatment / 5 / Control / 0.001 ± 0.001
(1 mmole/L, 10-15 min) / Pinacidil (50 mmole/L) / 0.493 ± 0.100
PdBu (50 nmole/L) + Pinacidil / 0.523 ± 0.111
6. Chelerythrine Pre-treatment / 5 / Control / 0.002 ± 0.001
(1 mmole/L, 10-15 min) / Pinacidil (50 mmole/L) / 0.727 ± 0.218
PdBu (50 nmole/L) + Pinacidil / 0.981 ± 0.308
* indicates significantly different from pinacidil alone by paired Student’s t test (P < 0.05)