Regulation of arterial tone by potassium channels.
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Animals↗
Biomedical subjects
M T Nelson
Publications and source records attributed to M T Nelson.
Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions.
The effects of tetraethylammonium ions (TEA+) and tetrapentylammonium ions (TPeA+) on Ca2(+) -activated K+ (KCa) channels were studied in membrane patches from mesenteric arterial myocytes. External TEA+ produced a flickery block. The concentration dependence for reduction in mean unitary current was consistent with 1:1 binding, with dissociation constants (Kd) in rat and rabbit of 196 and 159 microM at 0 mV, and the block was weakly voltage dependent. Rate constants for blocking and unblocking were 380 mM-1.ms-1 and 73 ms-1, respectively. In patches containing several channels TEA+ reduced average current to the same extent as mean unitary current, implying that TEA+ block is independent of the channel state. Block was unaffected by raising external K+ to 120 mM. External TPeA+ blocked with slower kinetics and lower affinity than TEA+ (Kd, 1.49 mM). The sulfonylurea glibenclamide (10-100 microM), the hyperpolarizing vasodilator cromakalim (5 microM), and internal ATP (1 mM) were without effect on channel activity. We conclude that TEA+ is a relatively effective blocker of single KCa channels of arterial smooth muscle and should block macroscopic currents equally well, whereas external TPeA+ is about eight times less effective.
Adenosine Triphosphate↗
Cromakalim and pinacidil dilate small mesenteric arteries but not small cerebral arteries.
Small elevations in external K+ hyperpolarize and dilate small cerebral arteries. The hyperpolarization and dilation to K+ are blocked by barium (less than 0.1 mM). Since membrane hyperpolarization appears to be an important mechanism for dilation of these small cerebral arteries, we investigated the effects of the hyperpolarizing vasodilators, cromakalim and pinacidil, on isolated pressurized rat cerebral arteries (diameter of 158 +/- 5 microns at 50% of the systolic blood pressure). Cromakalim and pinacidil, which are potent relaxants of a variety of muscle types, were without effect on posterior cerebral arteries at concentrations that completely dilate similarly sized rat mesenteric arteries (diameter 134 +/- 6 microns at 50% of the systolic blood pressure). The mesenteric artery dilation to cromakalim and pinacidil was reversed by glibenclamide. However, unlike the cerebral arteries, mesenteric arteries did not exhibit a barium-sensitive dilation to external K+. Thus it appears that there may be differences in the types of K+ channels that are activated by dilating mechanisms in small cerebral and mesenteric arteries.
Animals↗
ATP-sensitive K+ channels from aortic smooth muscle incorporated into planar lipid bilayers.
Glibenclamide binding sites were identified in a membrane preparation from canine aortic smooth muscle. The dissociation constant for [3H]glibenclamide binding was 10 +/- 2 nM, with a density of 420 +/- 108 fmol/mg protein. The properties of ATP-sensitive potassium (KATP) channels from the same membrane preparation incorporated into planar lipid bilayers were investigated. ATP was a potent inhibitor of the channels with half-maximal inhibition of channel activity by 41 microM ATP. Glibenclamide inhibited channel activity, and cromakalim activated the channel in the presence of ATP. Blockers of Ca(2+)-activated K+ (KCa) channels (charybdotoxin and tetraethylammonium ions) did not affect KATP channels in concentrations that caused significant block of KCa channels in bilayers. This membrane preparation should allow further biochemical and functional characterization of KATP channels and glibenclamide receptors in arterial smooth muscle.
Adenosine Triphosphate↗
Regulation of single calcium channels in cerebral arteries by voltage, serotonin, and dihydropyridines.
Unitary currents through Ca channels were measured from cell-attached patches on smooth muscle cells isolated from rabbit cerebral (basilar) arteries. Barium (80 and 10 mM) and calcium (80 and 10 mM) were used as the charge carriers. The dihydropyridine Ca channel agonist BAY R 5417 was used to increase open-state probability (Popen), with 500 nM BAY R 5417 increasing Popen 10-fold at 0 mV. Barium currents through single Ca channels were greater than calcium currents at any voltage, with single-channel conductances negative to -20 mV of 24.6 pS (80 mM barium), 15.1 pS (80 mM calcium), 17.2 pS (10 mM barium), and 5.8 pS (10 mM calcium). The single-channel Popen increased 2.7-fold per 5- to 7-mV membrane depolarization (negative to 0 mV) and was half maximal at +0.4 mV (80 mM calcium) and +13.5 mV (80 mM barium). Ca channels with calcium but not with barium as the charge carrier exhibited pronounced inactivation positive to -20 mV (half time, 112 ms at 0 mV). The dihydropyridine nimodipine (2 nM) inhibited average currents through Ca channels. The cerebral artery constrictor serotonin increased Popen of single Ca channels by as much as 200-fold without an effect on single-channel conductance. A second distinct amplitude of unitary currents was often observed, corresponding to a channel conductance of about one-half the more commonly observed level. The small-conductance-level channel was voltage dependent, did not inactivate over 0.5-s test pulses (with barium), and could be activated by serotonin.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Role of potassium channels in the vascular response to endogenous and pharmacological vasodilators.
Many endogenous and pharmacological vasodilators hyperpolarize vascular smooth muscle and this response appears to be due to an increased conductance to potassium ions. The hyperpolarization may contribute to the mechanism of dilation by causing voltage-dependent calcium channels to close. Recent evidence indicates that the response to hyperpolarizing vasodilators is mediated through activation of ATP-sensitive potassium (KATP) channels. Single KATP channels on isolated vascular smooth muscle cells are activated by cromakalim and calcitonin gene-related peptide (CGRP). This response is inhibited by glibenclamide. Cromakalim, CGRP and other vasodilators hyperpolarize and relax arteries in vitro and these responses are reversed by glibenclamide. The hypotensive effects of these agents in vivo are antagonized by glibenclamide. We propose that activation of KATP channels and the associated membrane hyperpolarization represents an important general mechanism of vasodilation.
Animals↗
Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels.
Calcitonin gene-related peptide (CGRP) is a 37-amino-acid peptide produced by alternative processing of messenger RNA from the calcitonin gene. CGRP is one of the most potent vasodilators known. It occurs in and is released from perivascular nerves and has been detected in the blood stream, suggesting that it is important in the control of blood flow. The mechanism by which it dilates arteries is not known. Here, we report that arterial dilations in response to CGRP are partially reversed by blockers of the ATP-sensitive potassium channel (K(ATP)), glibenclamide and barium. We also show that CGRP hyperpolarizes arterial smooth muscle and that blockers of K(ATP) channels reverse this hyperpolarization. Finally, we show that CGRP opens single K+ channels in patches on single smooth muscle cells from the same arteries. We propose that activation of K(ATP) channels underlies a substantial part of the relaxation produced by CGRP.
Adenosine Triphosphate↗
Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone.
Resistance arteries exist in a maintained contracted state from which they can dilate or constrict depending on need. In many cases, these arteries constrict to membrane depolarization and dilate to membrane hyperpolarization and Ca-channel blockers. We discuss recent information on the regulation of arterial smooth muscle voltage-dependent Ca channels by membrane potential and vasoconstrictors and on the regulation of membrane potential and K channels by vasodilators. We show that voltage-dependent Ca channels in the steady state can be open and very sensitive to membrane potential changes in a range that occurs in resistance arteries with tone. Many synthetic and endogenous vasodilators act, at least in part, through membrane hyperpolarization caused by opening K channels. We discuss evidence that these vasodilators act on a common target, the ATP-sensitive K (KATP) channel that is inhibited by sulfonylurea drugs. We propose the following hypotheses that presently explain these findings: 1) arterial smooth muscle tone is regulated by membrane potential primarily through the voltage dependence of Ca channels; 2) many vasoconstrictors act, in part, by opening voltage-dependent Ca channels through membrane depolarization and activation by second messengers; and 3) many vasodilators work, in part, through membrane hyperpolarization caused by KATP channel activation.
Animals↗
Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle.
Vasodilators are used clinically for the treatment of hypertension and heart failure. The effects of some vasodilators seem to be mediated by membrane hyperpolarization. The molecular basis of this hyperpolarization has been investigated by examining the properties of single K+ channels in arterial smooth muscle cells. The presence of adenosine triphosphate (ATP)-sensitive K+ channels in these cells was demonstrated at the single channel level. These channels were opened by the hyperpolarizing vasodilator cromakalim and inhibited by the ATP-sensitive K+ channel blocker glibenclamide. Furthermore, in arterial rings the vasorelaxing actions of the drugs diazoxide, cromakalim, and pinacidil and the hyperpolarizing actions of vasoactive intestinal polypeptide and acetylcholine were blocked by inhibitors of the ATP-sensitive K+ channels, suggesting that all these agents may act through a common pathway in smooth muscle by opening ATP-sensitive K+ channels.
Acetylcholine↗
External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.
The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Dihydropyridine inhibition of single calcium channels and contraction in rabbit mesenteric artery depends on voltage.
1. The effects of membrane potential and the dihydropyridine calcium channel inhibitor, nisoldipine, on single calcium channels in the presence of Bay K 8644 and contraction in the presence and absence of Bay K 8644 were examined in the rabbit mesenteric artery. 2. Membrane depolarization decreased the peak average single calcium channel current that could be elicited by a test pulse to 0 mV. The steady-state inactivation relationship could be described by the Boltzmann equation, [1 + exp[Vm-V0.5)/k)]-1, with a steepness factor, k, of 7.1 mV. Nisoldipine shifted the steady-state inactivation curve to more negative potentials by increasing the fraction of test pulses without openings. 3. The degree of nisoldipine inhibition of average single calcium channel currents increased with membrane depolarization. Depolarization of the holding potential from -100 to -55 mV decreased the concentration of nisoldipine needed for 50% inhibition (Kapp) from 12.1 to 1.9 nM in the presence of 1 microM-Bay K 8644. 4. Membrane depolarization by external potassium (K+) of the intact artery in the presence of nisoldipine decreased contractions evoked by depolarizing test pulses. The relationship between membrane potential and contraction could be empirically described by the Boltzmann equation, with a steepness factor, k, of 7.1 mV. Increasing the nisoldipine concentration from 0.25 to 2.0 nM shifted the mid-point of this relationship from -20.5 to -33.0 mV, without affecting the steepness factor. 5. Nisoldipine inhibition of contraction increased with membrane depolarization. Membrane depolarization from -68.6 to -30.0 mV decreased the Kapp of nisoldipine for contractions from 3.02 to 0.69 nM. Bay K 8644 (1 microM) elevated Kapp about 9.3-fold at 5 mM-K+. In the presence of Bay K 8644, membrane depolarization from -68.6 to -30.0 mV reduced Kapp from 28.4 to 4.0 nM. 6. In the presence of nisoldipine, the effect of membrane depolarization on the time course of development of inhibition was examined. In 3 nM-nisoldipine, after membrane depolarization with 20 mM-K+, the time course of development of inhibition of force could be described by a single exponential with a time constant of 16.5 min. Membrane depolarization to a more positive potential accelerated the development of inhibition. 7. The results were interpreted by a model in which nisoldipine binds with higher affinity to the inactivated state than to the resting state of calcium channels in the mesenteric artery. The approach presented here can be used to estimate the properties of steady-state calcium channel inactivation and dihydropyridine interactions in smooth muscle cells in the intact artery under physiological conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Phospholamban forms Ca2+-selective channels in lipid bilayers.
Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. A role for phospholamban in the control of Ca2+ transport by the sarcoplasmic reticulum has been postulated, but the mechanism is incompletely understood. Structural characterization of the purified protein suggests that it is capable of forming a membrane-spanning pore (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). The experiments described here tested the hypothesis that canine cardiac phospholamban, isolated in the fully dephosphorylated state, forms ion channels in lipid bilayers. Phospholamban purified by two different methods formed channels that were permeable to cations, exhibited spontaneous openings and closings, and were selective for Ca2+ over K+. Dihydropyridine drugs and ryanodine did not affect channel activity. The putative membrane-spanning portion of the molecule, residues 26-52, also formed channels in the bilayer. The putative regulatory portion of the molecule, residues 2-25, did not. The results suggest that phospholamban may regulate sarcoplasmic reticulum Ca2+ flux by acting as a Ca2+ channel.
Adenosine Triphosphatases↗
Noradrenaline contracts arteries by activating voltage-dependent calcium channels.
Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.
Animals↗
Calcium channels in thrombin-activated human platelet membrane.
Platelet-activating factor, 5-hydroxytryptamine, thromboxane A2, adenosine diphosphate and thrombin are known to activate platelets by stimulating calcium entry, but the nature of the entry pathways is unknown. We present the identification of single divalent cation channels from thrombin-activated human platelets. Membrane vesicles from unstimulated and thrombin-stimulated human platelets were incorporated in planar bilayers and unitary currents through single channels were measured. Divalent cation selective channels could only be demonstrated in thrombin-stimulated preparations. These channels share a number of properties in common with voltage-dependent calcium channels--a high degree of selectivity for divalent cations, a single channel conductance of about 10 pS (in 150 mM Ba2+) and sensitivity to blockade by inorganic calcium channel blockers such as Ni2+. In other respects, these channels are different as they are not voltage-dependent and are not blocked by 1,4-dihydropyridine calcium channel antagonists.
Barium↗
Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.
Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.
Animals↗
Effects of permeant ions and blockers on properties of single calcium channels from brains.
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Animals↗
Effects of the ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump.
(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. In the reconstituted system the pump can be activated by adding ATP to the external medium. ATP-driven potassium extrusion by the Na+,K+-pump was studied using a voltage-sensitive dye in the presence of valinomycin. ADP strongly reduced the turnover rate of the pump with a concentration for half-maximal inhibition of cD,1/2 = 0.1 mM. cD,1/2 was found to be virtually independent of ATP concentration, indicating that the inhibition is non-competitive with respect to ATP. The non-competitive inhibition by ADP can be explained on the basis of the Post-Albers reaction cycle of the Na+,K+-pump, assuming that the main action of ADP is the reversal of the phosphorylation step. A similar 'product inhibition' was observed with inorganic phosphate, but at much higher concentrations (cP,1/2 = 14 mM).
Adenosine Diphosphate↗
Single nisoldipine-sensitive calcium channels in smooth muscle cells isolated from rabbit mesenteric artery.
Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.
Animals↗