1. Endothelium-derived relaxing factors (EDRF), including nitric oxide (NO), prostacyclin (PGI2) and an as yet uncharacterized endothelium-derived hyperpolarizing factor (EDHF), are now recognized to induce relaxation of vascular smooth muscle, in part via the activation of K+ channels. 2. Experiments using selective K+ channel blockers, including iberiotoxin (IbTX), glibenclamide, apamin and 4-aminopyridine (4-AP) to inhibit endothelium-induced relaxation suggest that more than one type of K+ channel may be involved, depending on the species and tissue, including: (i) large conductance Ca(2+)-activated (BKCa) channels; (ii) ATP-sensitive (KATP) channels; (iii) small conductance Ca(2+)-activated (SKCa) channels; and (iv) voltage-gated (Kv) K+ channels. 3. Recent observations suggest a role for Kv channels in some vessels based on a sensitivity of NO- and PGI2-mediated relaxations to 4-AP, as well as a complete suppression of EDHF-dependent relaxation by a combination of charybdotoxin (ChTX) and apamin but not IbTX and apamin. 4. The molecular identity of the K+ channels affected by EDRF is not well characterized. Recently, findings indicate that the pore-forming alpha-subunit tetramers of vascular smooth muscle BKCa channels are due to the expression of the so-called Slo channel gene. The identities of the KATP, SKCa and Kv channels involved in endothelium-dependent vasodilation are not known. 5. The component of whole-cell Kv current affected by PGI2 may be due to slowly inactivating, 4-AP-sensitive, 15 pS delayed-rectifier K+ channels (KDR); the activity of these channels in vascular myocytes is increased by forskolin and protein kinase A (PKA) and rabbit portal vein Kv1.5 pore-forming alpha-subunits, which appear to be a component of native KDR current and possess consensus phosphorylation sequences for PKA.

1. The effect of angiotensin II (Ang) on delayed rectifier K+ current (IK(v)) was studied in isolated rabbit portal vein smooth muscle cells using standard whole-cell voltage clamp technique. The effect of 100 nM Ang on macroscopic, whole-cell IK(V) was assessed in myocytes dialysed with 10 mm BAPTA, 5 mm ATP and 1 mm GTP either at room temperature or at 30 'C. 2. Application of Ang caused a decline in IK(V) which was reversed upon washout of the drug.Tail current recorded after 250 ms pulses to +30 mV and repolarization to -40 mV was reduced from 3'9 + 0 7 to 2-5 + 0 5 pA pF-' at 20 'C (n = 6) and from 4-5 + 0 5 to 3-13 + 04 pA pF-1 at 30 C (n = 17). 3. Ang had no effect on outward current in the presence of an AT1 selective antagonist, losartan (1 uM), which alone had no direct effect on the amplitude of IK(v). Substitution of extracellular Ca2+ with Mg2+ in the presence of 10 mm intracellular BAPTA did not affect the suppression of IK(V) by Ang. 4. Ang induced a decrease in time constant for the rapid phase of inactivation of the macroscopic current (r1 reduced from 377 + 32 to 245 + 11 ms; T2 unchanged, n = 17).Neither the voltage dependence of activation nor inactivation were affected by Ang. The inhibition of IK(V) by Ang was abolished by intracellular dialysis with the selective PKCinhibitors, calphostin C (1 IBM) and chelerythrine (50 uM). These data provide strong evidence that the decline in IK(V) due to Ang treatment is due to PKC activation. 6. The pattern of expression of PKC isoforms was examined in rabbit portal vein using isoenzyme-specific antibodies: a, e and ; isoenzymes were detected, but /1, y, a and V isoenzymes were not.7. The lack of requirement for Ca2+, as well as the sensitivity of the Ang response to chelerythrine, suggest the involvement of the Ca2P-independent PKC isoenzyme e in the signal transduction pathway responsible for IK(V) inhibition by Ang.Variations in intracellular Ca2+ concentration constitute an important element in the control of vascular smooth muscle tone. Contractile agonists are known to cause an elevation in intracellular Ca2+ concentration by increasing the influx of extracellular Ca2P through voltage-dependent L-type Ca2P channels and/or by releasing Ca2+ from intracellular stores.

Quebec, Canada HiT 1C81. We studied the biophysical properties of single large conductance (>200 pS in symmetrical K+ pipette and bath solutions) Ca2P-activated K+ (BKCa) channels of rabbit portal vein and coronary arterial smooth muscle cells using the cell-attached and inside-out variants of the patch-clamp technique (at 22 'C).

Acetylcholine (ACh) increases the amplitude and duration of colonic electrical slow waves. This suggests that ACh either increases an inward current or suppresses an outward current. The latter hypothesis was tested in whole cell voltage-clamp experiments performed on freshly dispersed smooth muscle cells from canine proximal colon. Addition of ACh (10(-5) M) to solutions bathing cells reduced time-dependent outward currents elicited by depolarizing test pulses in the range of -45 to +30 mV. Analysis of tail currents showed that ACh caused a 10- to 15-mV positive shift in voltage-dependent activation. When cells were pretreated with 10(-6) M nifedipine to abolish the Ca2+-dependent component of the outward current, the reduction of outward current by ACh was blocked. Single-channel experiments were performed to determine whether ACh had a direct effect on Ca2+-activated K channels. ACh, 10(-5) M, added to bath and pipette solutions caused a positive shift in voltage-dependent activation in on-cell experiments. This effect of ACh on Ca2+-activated K channels provides a mechanism for the effects of muscarinic, excitatory stimulation of circular muscle of the colon.

Outward currents of colonic smooth muscle cells were characterized by the whole cell voltage-clamp method. Four components of outward current were identified: a time-independent and three time-dependent components. The time-dependent current showed strong outward rectification positive to -25 mV and was blocked by tetraethylammonium. The time-dependent components were separated on the basis of their time courses, voltage dependence, and pharmacological sensitivities. They are as follows. 1) A Ca2+-activated K current sensitive to external Ca2+ and Ca2+ influx was blocked by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.1 X 10(-3) M) and nifedipine (1 X 10(-6) and was increased by elevated Ca2+ (8 X 10(-6) M) and BAY K 8644 (1 X 10(-6) M). 2) A "delayed rectifier" current was observed that decayed slowly with time and showed no voltage-dependent inactivation. 3) Spontaneous transient outward currents that were blocked by ryanodine (2 X 10(-6) M) were also recorded. The possible contributions of these currents to the electrical activity of colonic muscle cells in situ are discussed. Ca2+-activated K current may contribute a significant conductance to the repolarizing phase of electrical slow waves.