Calcium and voltage dependence of single Ca2+-activated K+ channels from cultured hippocampal neurons of rat

Calcium and voltage dependence of the Ca2+-activated K+ channel, K(Ca), was studied at the single-channel level in cultured hippocampal neurons from rat. The K(Ca) channel has approx. 220 pS conductance in symmetrical 150 mM K+, and is gated both by voltage and by Ca2+ ions. For a fixed Ca2+ concentration at the inner membrane surface, [Ca]i, channel open probability, Po, increases e-fold for 14 mV positive change in membrane potential. At a fixed membrane potential (0 mV), channel activity is first observed at [Ca]i = 10(-6) M, and increases with Ca2+ concentration approximating an absorption isotherm with power 1.4. The [Ca]i required to half activate (Po = 0.5) the channel is 4.10(-6) M. When compared to other preparations, the K(Ca) channel from hippocampal neurons reported here shows the lowest Ca2+ sensitivity and the highest voltage sensitivity. These findings are interpreted in evolutionary terms.     

Membrane-Delimited Phosphorylation Enables the Activation of the Outward-Rectifying K Channels in Motor Cell Protoplasts of Samanea saman

Outward-rectifying K channels activated by membrane depolarization (Kout or KD channels) control K+ efflux from plant cells. To find out to what extent phosphorylation is required for the activity of these channels, the patch-clamp method was applied to protoplasts from the legume Samanea saman in both whole-cell and isolated-patch configurations. In the absence of either Mg2+ or ATP in the "cytosolic" solution, the KD channel activity declined completely within 15 min. This decline could be reversed in excised, inside-out patches by restoring MgATP (1 mM) to the cytoplasmic side of the membrane. Mg2+ (1 mM) plus 5[prime]-adenylylimidodiphosphate (1 mM), a nonhydrolyzable ATP analog, did not substitute for ATP. Mg2+ (1 mM) plus adenosine 5[prime]-O-(3-thiotriphosphate) (25 to <100 [mu]M), an irreversibly thiophosphorylating ATP analog, sustained channel activity irreversibly. 1-(5-IsoquinolinesulphonyI)-2- methylpiperazine (100 [mu]M), a broad-range kinase inhibitor, blocked the activity of KD channels in the presence of MgATP. These results strongly suggest that the activation of the outward-rectifying K channels by depolarization depends critically on phosphorylation by a kinase tightly associated with the KD channel.     

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new type of amiloride-sensitive cationic channel in endothelial cells of brain microvessels

Endothelial cells from brain microvessels form the blood-brain barrier. Brain microvessels and endothelial cells isolated from rat brain microvessels express an amiloride-sensitive cationic channel that was characterized using [3H]phenamil binding and patch-clamp experiments. [3H]Phenamil, a labeled amiloride analog, recognizes a single family of binding sites with a dissociation constant of 20-30 nM and a maximum binding capacity of 8-15 pmol/mg protein. The pharmacological profile of the channel (phenamil greater than benzamil greater than amiloride) is very similar to that of the epithelium Na+ channel of mammalian kidney and of frog epithelia. Long-lasting currents were observed in patch-clamp experiments using excised outside-out patches. Application of amiloride or phenamil first produced a rapid flickering of channel activity and then its complete blockade. The mean unit channel conductance at 140 mM Na+ was 23 picosiemens. The selectivity of Na+ over K+ was estimated from reversal potentials to be 1.5:1. Properties of the channel in microvessels are clearly distinct from those of the Na+ channel of the kidney, suggesting the existence of several isoforms of cationic channels that are sensitive to amiloride and its derivatives. The low selectivity cationic channel of endothelial cells in brain microvessels might be important for controlling both Na+ and K+ movements across the blood-brain barrier.     

Calcium channel activation in vascular smooth muscle by BAY K 8644

BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate) and CGP 28 392 (ethyl-4(2-difluoromethoxyphenyl)-1,4,5,7-tetrahydro-2-methyl-5-++ +oxofuro- [3,4-b]pyridine-3-carboxylate) are closely related in structure to nifedipine and other 1,4-dihydropyridine Ca2+ channel antagonists. However, both BAY K 8644 and CGP 28 392 serve as activators of Ca2+ channels. In the rat tail artery, responses to BAY K 8644 are dependent upon Ca2+ext and prior stimulation by K+ or by the alpha-adrenoceptor agonists, phenylephrine and BHT 920 (6-allyl-2-amino-5,6,7,8,-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). Responses are blocked noncompetitively by the Ca2+ channel antagonists D-600 [-)-D-600 greater than (+)-D-600) and diltiazem, but competitively by nifedipine (pA2 = 8.27). This suggests that activator and inhibitor 1,4-dihydropyridines interact at the same site. BAY K 8644 potentiates K+ responses and Ca2+ responses in K+-depolarizing media. The leftward shift of the K+ dose--response curve produced by BAY K 8644 suggests that this ligand facilitates the voltage-dependent activation of the Ca2+ channel. The pA2 value for nifedipine antagonism of BAY K 8644 responses is significantly lower than that for nifedipine antagonism of Ca2+ responses in K+ (25-80 mM) depolarizing media (9.4-9.6), suggesting that the state of the channel may differ according to the activating stimulus.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Purified calcium channels have three allosterically coupled drug receptors

(-)-[3H]Desmethoxyverapamil and (+)-[3H]PN 200-110 were employed to characterize phenylalkylamine-selective and 1,4-dihydropyridine-selective receptors on purified Ca2+ channels from guinea-pig skeletal muscle t-tubules. In contrast to the membrane-bound Ca2+ channel, d-cis-diltiazem (EC50 = 4.5 +/- 1.7 microM) markedly stimulated the binding of (+)-[3H]PN 200-110 to the purified ionic pore. In the presence of 100 microM d-cis-diltiazem (which binds to the benzothiazepine-selective receptors) the Bmax for (+)-[3H]PN 200-110 increased from 497 +/- 81 to 1557 +/- 43 pmol per mg protein, whereas the Kd decreased from 8.8 +/- 1.7 to 4.7 +/- 1.8 nM at 25 degrees C. P-cis-Diltiazem was inactive. (-)-Desmethoxyverapamil, which is a negative heterotropic allosteric inhibitor of (+)-[3H]IN 200-110 binding to membrane-bound channels, stimulated 1,4-dihydropyridine binding to the isolated channel. (-)-[3H]Desmethoxyverapamil binding was stimulated by antagonistic 1,4-dihydropyridines [(+)-PN 200-110 greater than (-)(R)-202-791 greater than (+)(4R)-Bay K 8644] whereas the agonistic enantiomers (+)(S)-202-791 and (-)(4S)-Bay K 8644 were inhibitory and (-)-PN 200-110 was inactive. The results indicate that three distinct drug-receptor sites exist on the purified Ca2+ channel, two of which are shown by direct labelling to be reciprocally allosterically coupled.     

Modulation of high affinity phenylalkylamine binding sites on cultured human embryonal vascular smooth muscle cells.

Two phenylalkylamine Ca2+ channel ligands, (+/-)-[3H]verapamil ((+/-)-[3H]V) (-)-[3H]desmethoxyverapamil ((-)-[3H]DV), were employed in whole cell binding assays to characterize the specific high affinity binding sites on Ca2+ channels, their cooperativity and modulations induced on cultured human embryonal vascular smooth muscle preparation (VSM) by: 1) Beta-adrenergic stimulation of the cell, 2) exposure to high K+ concentration, 3) exposure to high concentration of Mg2+ ions, 4) the presence of a benzothiazepine Ca2+ channel antagonist and modulator d-cis-diltiazem, and 5) guanylylimidodiphosphate. The total amounts of specific (+/-)-[3H]V and (-)-[3H]DV binding sites present on VSM cells increased significantly after beta-adrenergic receptor activation, following cell membrane depolarization induced by high concentrations of K+, in the presence of Ca2+ chelator Na3EDTA, and after incubation of VSM cells with a benzothiazine-type Ca2+ channel blocker d-cis-diltiazem. A marked reduction of (-)-[3H]DV binding was observed after permanent G-protein activation by a nonhydrolyzable analog of guanylylimidodiphosphate, after incubation of the cells with norepinephrine, and after incubation of VSM cells with millimolar concentration of Mg2+. The results suggest the existence of multiple modulations of specific (-)-[3H]DV binding sites on Ca2+ channel corresponding to the way of activation of the cell and also to the immediate "state" of the membrane bound Ca2+ channels present on VSM cells, the positive heterotropic interaction after beta-adrenergic stimulation, the homotropic positive allosteric interaction induced by d-cis-diltiazem and pure noncompetitive inhibition induced by guanylylimidodiphosphate. The presence of high concentrations of Mg2+ inhibited whereas the presence of Ca2+ chelator, of ethylenediamine-tetraacetic acid sodium salt, significantly increased the total number of specific high affinity (-)-[3H]DV binding sites on VSM cells.     

Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes

Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/-)[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half-maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and (-)D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM).     

Access channel model for the voltage dependence of the forward-running Na+/K+ pump

The voltage dependence of steady state current produced by the forward mode of operation of the endogenous electrogenic Na+/K+ pump in Na(+)- loaded Xenopus oocytes has been examined using a two-microelectrode voltage clamp technique. Four experimental cases (in a total of 18 different experimental conditions) were explored: variation of external [Na+] ([Na]o) at saturating (10 mM) external [K+] ([K]o), and activation of pump current by various [K]o at 0, 15, and 120 mM [Na]o (tetramethylammonium replacement). Ionic current through K+ channels was blocked by Ba2+ (5 mM) and tetraethylammonium (20 mM), thereby allowing pump-mediated current to be measured by addition or removal of external K+. Control measurements and corrections were made for pump current run-down and holding current drift. Additional controls were done to estimate the magnitude of the inwardly directed pump-mediated current that was present in K(+)-free solution and the residual K(+)- channel current. A pseudo two-state access channel model is described in the Appendix in which only the pseudo first-order rate coefficients for binding of external Na+ and K+ are assumed to be voltage dependent and all transitions between states in the Na+/K+ pump cycle are assumed to be voltage independent. Any three-state or higher order model with only two oppositely directed voltage-dependent rate coefficients can be reduced to an equivalent pseudo two-state model. The steady state current-voltage (I-V) equations derived from the model for each case were simultaneously fit to the I-V data for all four experimental cases and yielded least-squares estimates of the model parameters. The apparent fractional depth of the external access channel for Na+ is 0.486 +/- 0.010; for K+ it is 0.256 +/- 0.009. The Hill coefficient for Na+ is 2.18 +/- 0.06, and the Hill coefficient for K+ (which is dependent on [Na]o) ranges from 0.581 +/- 0.019 to 1.35 +/- 0.034 for 0 and 120 mM [Na]o, respectively. The model provides a reasonable fit to the data and supports the hypothesis that under conditions of saturating internal [Na+], the principal voltage dependence of the Na+/K+ pump cycle is a consequence of the existence of an external high- field access channel in the pump molecule through which Na+ and K+ ions must pass in order to reach their binding sites.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Calcium ions inhibit the allosteric interaction between the dihydropyridine and phenylalkylamine binding site on the voltage-gated calcium channel in heart sarcolemma but not in skeletal muscle transverse tubules

Calcium channel blockers bind with high affinity to sites on the voltage-sensitive Ca2+ channel. Radioligand binding studies with various Ca2+ channel blockers have facilitated identification and characterization of binding sites on the channel structure. In the present study we evaluated the relationship between the binding sites for the Ca2+ channel blockers on the voltage-sensitive Ca2+ channel from rabbit heart sarcolemma and rabbit skeletal muscle transverse tubules. [3H]PN200-110 binds with high affinity to a single population of sites on the voltage-sensitive Ca2+ channel in both rabbit heart sarcolemma and skeletal muscle transverse tubules. [3H]PN200-110 binding was not affected by added Ca2+ whereas EGTA and EDTA noncompetitively inhibited binding in both types of membrane preparations. EDTA was a more potent inhibitor of [3H]PN200-110 binding than EGTA. Diltiazem stimulates the binding of [3H]PN200-110 in a temperature-sensitive manner. Verapamil inhibited binding of [3H]PN200-110 to both types of membrane preparations in a negative manner, although this effect was of a complex nature in skeletal muscle transverse tubules. The negative effect of verapamil on [3H]PN200-110 binding in cardiac muscle was completely reversed by Ca2+. On the other hand, Ca2+ was without effect on the negative cooperativity seen between verapamil and [3H]PN200-110 binding in skeletal muscle transverse tubules. Since Ca2+ did not affect [3H]PN200-110 binding to membranes, we would like to suggest that Ca2+ is modulating the negative effect of verapamil on [3H]PN200-110 binding through a distinct Ca2+ binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

''Molten-globule'' state accumulates in carbonic anhydrase folding

Binding characteristics of [3H]BAY K 8644, a new class of pharmacologically potent compounds, the calcium channel activating dihydropyridines (DHP), were demonstrated in cultured myocardial cells. [3H]BAY K 8644 exhibited reversible and saturable binding to myocytes, and specific binding was Ca2+-dependent. The equilibrium dissociation constant, Kd, was 35.2 nM, and maximal binding capacity, Bmax, was 1.07 pmol/mg protein. Binding of the 3H-ligand was highly specific for various potently displacing DHP derivatives (either the calcium channel activating BAY K 8644, or the Ca2+ entry blockers of the nifedipine type) with inhibition constants (Ki values) in the nanomolar range. BAY K 8644, on the other hand, showed very low affinity to other receptors tested in brain and heart membranes. Displacement potency of BAY K 8644 correlated well with data of the functional pharmacology; e.g., the enhanced myocardial contractility. Results from competition studies using [3H]BAY K 8644 and [3H]nimodipine support the conclusion that both the channel activating and inhibiting DHP structures interact with the same specific receptor site that might be associated with the putative Ca2+-channel.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.     

The Gibbs-Donnan near-equilibrium system of heart

The gradients of the major inorganic ions across the plasma membrane of heart were examined to determine the factors controlling the extent and direction of the changes induced during injury, certain diseases, and electrolyte disturbances. The ionic environment was altered by changing only the concentration of inorganic phosphate, [sigma Pi]o, from 0 to 1.2 to 5 mM in the Krebs-Henseleit buffer perfusing working rat hearts. Raising [sigma Pi]o from 1.2 to 5 mM resulted in a decrease in total Mg2+ content and calculated free cytosolic [Mg2+] from 0.44 to 0.04 mM, conversion of 4 mmol of MgATP2- to ATP4- and a decrease in measured intracellular [Cl-]i from 41 to 16 mM. At all levels of [sigma Pi]o, both the [Na+]i and [K+]i were invariant at about 3 mM and 130 mM, respectively, as was the energy of hydrolysis of the terminal phosphate bond of sigma ATP, delta GATP Hydr, of -13.2 kcal/mol. The relationship maintained between the ions on both sides of the plasma membrane by the 3Na+/2K(+)transporting ATPase (EC 3.6.1.37) and an open K+ channel was: (formula; see text) The energy of the gradients of the other inorganic ions across the plasma membrane, delta G[ion]o/i, exhibited three distinct quanta of energy derived from the prime quantum of delta GATP Hydr of -13.2 kcal/mol. The second quantum was about one-third of delta GATP Hydr or +/- 4.4 kcal/mol and comprised the delta G[Na+]o/i, delta G[Mg2+]o/i, and delta G[HPO42-]o/i. These results indicated near-equilibrium was achieved by the reactants of the 3Na+/2K(+)-ATPase, the K+ channel, the Na(+)-Pi co-transporter, and a postulated net Mg2+/H2PO4- exchanger. The third quantum was one-third of delta G[Na+]o/i or about +/- 1.5 kcal/mol and comprised delta G[H+]o/i, delta G[HCO3-]o/i, and delta G[Cl-]o/i. The delta G[K+]o/i was 0, indicating near-equilibrium between the chemical energy of [K+]o/i and the E across the plasma membrane of -83 mV. It is concluded that the gradients of the major inorganic ions across the plasma membrane and the potential across that membrane constitute a Gibbs-Donnan equilibrium system catalyzed by transport enzymes sharing common substrates. The chemical and electrical energies of those gradients are equal in magnitude and opposite in sign to the chemical energy of ATP hydrolysis.     

Comparative Effects of Chronic Exposure to Ethanol and Calcium Channel Antagonists on Calcium Channel Antagonist Receptors in Cultured Neural (PC12) Cells

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.     

Effects of Antidepressant Drug Treatments on α2-Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes

KCl (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+-dependent manner; this release was attenuated by clonidine (0.01-100 microM). Changes in the release of [3H]NA and the functional status of alpha 2-adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 microM) or the potentiating effects of yohimbine (1 microM) on K+-evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous + K+-evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+-evoked release from the DMI-treated tissue. Attenuation of K+-evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI-treated tissue remained significantly reduced although total percent and K+-evoked [3H]NA release were unchanged. The K+-evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+-evoked [3H]NA release by clonidine in both chronic antidepressant-treated tissues was not significantly changed. It is concluded that the functional sensitivity of alpha 2-adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+-evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants.(ABSTRACT TRUNCATED AT 250 WORDS)