Age-related features of the effects of alpha- and beta-adrenomimetics on the glucocorticoid function of the adrenal glands]

In vitro effect of alpha-adrenomimetic naphtizin (0.1 mM X 1(-1) and beta-adrenomimetic isoproterenol (10 mcM X 1(-1) on the basal and ACTH-stimulated (10 U X 1(-1) production of II-oxoketosteroids (II-OKS) by isolated adrenals (IA) was studied in adult (6 mo) and old (28 mo) male rats. Naphtizin increased ACTH-stimulated secretion of II-OKS by IA of adult rats, but had no effect on the above secretion of old rats. Isoproterenol enhanced basal secretion of II-OKS by adult IA. No such effect on IA was found in old rats. There was no additive effect of isoproterenol on ACTH-stimulated II-OKS secretion by IA in both age groups.     

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Delayed rectifier potassium channels contribute to the depressed pulmonary artery contractility in pneumonia.

We investigated the role of K(+) channels in the attenuated pulmonary artery (PA) contractility characteristic of acute Pseudomonas pneumonia. Contractility of PA rings from the lungs of control or pneumonia rats was assessed in vitro by obtaining cumulative concentration-response curves to the contractile agonists KCl, phenylephrine, or PGF(2 alpha) on PA rings before and after treatment with K(+) channel blockers. In rings from pneumonia rats, paxilline (10 microM), tetraethylammonium (2 mM) (blockers of large-conductance Ca(2+)-activated K(+) channels), and glybenclamide (ATP-sensitive K(+) channel blocker, 80 microM) had no significant effect on the attenuated contractile responses to KCl, phenylephrine, and PGF(2 alpha). However, 4-aminopyridine (2 mM), a blocker of voltage-gated K(+) channels (delayed rectifier K(+) channel) reversed this depressed contractility. Therefore, large-conductance Ca(2+)-activated K(+) and ATP-sensitive K(+) channels do not contribute to the attenuated PA contractility observed in this model of acute pneumonia. In contrast, 4-aminopyridine enhances contraction in PA rings from pneumonia lungs, consistent with involvement of a voltage-gated K(+) channel in the depressed PA contractility in acute pneumonia. Unraveling the precise mechanism of attenuated contractility in pneumonia could lead to innovative therapies for the pulmonary vascular abnormalities associated with this disease.     

Nicardipine as a Ca2+ channel blocker in single barnacle muscle fibers

A study has been made of the efficacy of nicardipine as a Ca2+ channel blocker by determining the magnitude of its effect on the stimulatory response of the ouabain-insensitive Na+ efflux in single barnacle muscle fibers to 100 mM external K+. The results show that nicardipine (at pH 6.5) is a potent inhibitor, the minimal effective concentration being approx. 10(-7) M and the IC(50) about 5.10(-6) M. Nicardipine, however, is not as potent as verapamil (at pH 6.5) on an equimolar basis. This is explained by assuming that the number of dihydropyridine receptors in the t-tubule membranes of barnacle fibers is not high or that verapamil is able to block the sarcoplasmic reticulum Ca2+ release channel in addition to the voltage-dependent Ca2+ channels.     

Paradoxical effects of K+ and D-600 on parathyroid hormone secretion and cytoplasmic Ca2+ in normal bovine and pathological human parathyroid cells

The effects of K+ and the Ca2+ channel blocker D-600 on parathyroid hormone (PTH) release and cytoplasmic Ca2+ activity (Ca2+i) were measured at different Ca2+ concentrations in dispersed parathyroid cells from normal cattle and from patients with hyperparathyroidism. When the extracellular Ca2+ concentration was raised within the 0.5-3.0 mM range Ca2+i increased and PTH secretion was inhibited. There was also a stimulatory effect of Ca2+ on secretion as indicated by a parallel decrease of Ca2+i and PTH release when extracellular Ca2+ was reduced to less than 25 nM. Addition of 30-50 mM K+ stimulated PTH release and lowered Ca2+i. The effect of K+ was less pronounced in the human cells with a decreased suppressability of PTH release. The Ca2+ channel blocker D-600 had no effect on Ca2+i and PTH release in the absence of extracellular Ca2+. However, at 0.5-1.0 mM Ca2+, D-600 increased Ca2+i and inhibited PTH release, whereas the opposite effects were obtained at 3.0 mM Ca2+. The transition from inhibition to stimulation occurred at a higher Ca2+ concentration in the human cells and the right-shift in the dose-effect relationship for Ca2+-inhibited PTH release tended to be normalized by D-600. It is suggested that K+ stimulates PTH release by increasing the intracellular sequestration of Ca2+ and that the reduced response in the parathyroid human cells is due to the fact that Ca2+i already is lowered. D-600 appears to have both Ca2+ agonistic and antagonistic actions in facilitating and inhibiting Ca2+ influx into the parathyroid cells at low and high concentrations of extracellular Ca2+, respectively. D-600 and related drugs are considered potentially important for the treatment of hyperparathyroidism.     

A forskolin and verapamil sensitive K+ current in human tracheal cells

A voltage-dependent K+ current has been revealed in whole-cell recordings carried out on immortalized cells obtained from the human tracheal epithelium. At positive membrane potentials the current shows a time dependent inactivation which is accelerated by increasing the depolarizing step. Forskolin, a direct activator of adenylyl cyclase, and verapamil, a Ca2+ channel blocker, induce the K+ current to inactivate more rapidly. Control experiments show that the action of these two compounds is not mediated by cyclic AMP and Ca2+. The application of 1,9-dideoxyforskolin, an analogue which does not stimulate adenylate cyclase, inhibits the current in the same way as forskolin; on the contrary, the dibutyryl analogue of cyclic AMP is ineffective. Furthermore, eliminating extracellular Ca2+ does not affect K+ current kinetics. Tetraethylammonium is an effective blocker of this current with an IC50 of 0.3 mM.     

Corticosteroid regulation of Na+ and K+ transport in the rat distal colon during postnatal development

To study the role of corticosteroids in the regulation of colonic electrogenic amiloride-sensitive Na+ absorption (ISCNa) and barium-sensitive K+ secretion (ISCK) during development, we investigated suckling (10-day old), weanling (25-day old) and adult (90-day old) adrenalectomized rats after they had received aldosterone, dexamethasone or corticosterone. Adrenalectomy reduced markedly ISCNa in suckling rats and completely inhibited ISCNa in weanling animals; the ISCNa was absent in intact adult rats. The doses of aldosterone, corticosterone and dexamethasone estimated to be equivalent to the endogenous production rate of aldosterone and corticosterone restored ISCNa after 1 day in both suckling and weanling rats. Compared with aldosterone, glucocorticoids produced a greater increase in ISCNa. Concurrent spironolactone treatment (a mineralocorticoid antagonist) completely prevented the effect of aldosterone but had no effect in dexamethasone-treated rats. The glucocorticoid antagonist RU 38 486 inhibited the dexamethasone-induction of ISCNa but had no effect on aldosterone. The response to corticosteroids, measured as the increase of ISCNa, declined from suckling to adult rats. In contrast to ISCNa, the same time of treatment and the same doses of corticosteroids did not influence ISCK. ISCK was stimulated only after chronic treatment (4 days). These findings suggest that, in the distal colon of young rats, (1) both corticosteroids may regulate amiloride-sensitive Na+ absorption and barium-sensitive K+ secretion, (2) different receptors mediate the colonic effects of glucocorticoids and mineralocorticoids, (3) immature rats are more sensitive to corticosteroids than adult animals, and (4) the acute effect of corticosteroids is an increase in Na+ absorption which is followed by delayed stimulation of K+ secretion.     

Voltage- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

Effects of extracellular calcium on canine tracheal smooth muscle

Strips of canine tracheal smooth muscle were studied in vitro to determine the effects of changes in the extracellular calcium (Cao) concentration on tonic contractions induced by acetylcholine and 5-hydroxytryptamine. Strips were contracted with graded concentrations of the above agents in 2.4 mM Ca, after which CaCl2 was administered to achieve final concentrations of 5.0, 10.0, and 20.0 mM. Increases in Cao to 5 mM or above caused significant relaxation of muscles contracted with 5-hydroxytryptamine but did not significantly relax muscles contracted with acetylcholine. Increases in Cao also caused significant relaxation of muscles contracted with low concentrations of K+ (20 or 30 mM). However, in 60 or 120 mM K+, increases in Cao resulted predominantly in muscle contraction. Inhibition of the Na+-K+-ATPase by ouabain (10(-5) M) or K+ depletion reversed the effects of Cao from relaxation to contraction in tissues contracted with 5-hydroxytryptamine. Increases in Cao also caused contraction rather than relaxation in the presence of verapamil (10(-6) M). We conclude that calcium has both excitatory and inhibitory effects on the contractile responses of canine tracheal smooth muscle. The inhibitory effects of Ca2+ appear to be linked to the activity of the membrane Na+-K+-ATPase.     

Diclofenac-induced peripheral antinociception is associated with ATP-sensitive K+ channels activation

In order to investigate to the contribution of K+ channels on the peripheral antinociception induced by diclofenac, we evaluated the effect of several K+ channel blockers, using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 microg) of prostaglandin E2. Diclofenac administered locally into the right hindpaw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. This blockade of PGE2 mechanical hyperalgesia induced by diclofenac (100 microg/paw) was antagonized in a dose-dependent manner by intraplantar administration of the sulphonylureas glibenclamide (40, 80 and 160 microg) and tolbutamide (80, 160 and 320 microg), specific blockers of ATP-sensitive K+ channels, and it was observed even when the hyperalgesic agent used was carrageenin, while the antinociceptive action of indomethacin (200 microg/paw), a typical cyclo-oxygenase inhibitor, over carrageenin-induced hyperalgesia was not affected by this treatment. Charybdotoxin (2 microg/paw), a blocker of large conductance Ca2+-activated K+ channels and dequalinium (50 microg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of diclofenac. This effect was also unaffected by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 microg) and 4-aminopyridine (100 microg) or cesium (500 microg), a non-specific K+ channel blocker. The peripheral antinociceptive effect induced by diclofenac was antagonized by NG-Nitro L-arginine (NOarg, 50 microg/paw), a NO synthase inhibitor and methylene blue (MB, 500 microg/paw), a guanylate cyclase inhibitor, and this antagonism was reversed by diazoxide (300 microg/paw), an ATP-sensitive K+ channel opener. We also suggest that an endogenous opioid system may not be involved since naloxone (50 microg/paw) did not affect diclofenac-induced antinociception in the PGE2-induced hyperalgesia model. This study provides evidence that the peripheral antinociceptive effect of diclofenac may result from activation of ATP-sensitive K+ channels, possible involving stimulation of L-arginine/NO/cGMP pathway, while Ca2+-activated K+ channels, voltage-dependent K+ channels as well as endogenous opioids appear not to be involved in the process.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Effect of calcium on corticosteroid secretion by isolated frog interrenal gland

The direct effect of extracellular calcium concentrations on corticosteroidogenesis has been examined in the frog, using a perifusion system technique. The release of corticosterone and aldosterone in the effluent medium was monitored by specific radioimmunoassays. Increasing concentrations of Ca2+ (from 2 to 15 mM) gave rise to a dose-related stimulation of corticosteroid release, whereas the increment of either Na+ or K+ concentrations did not modify steroid production. Iterative administration of a moderate concentration of calcium (6 mM) led to a reproducible stimulation of steroid secretion whereas the same dose infused during 6 h induced a transient rise in corticosteroid secretion followed by a plateau. The direct effect of Ca2+ on steroidogenesis was confirmed by the dose-dependent stimulation of steroid secretion induced by the calcium ionophore A 23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mM Co2+ both resulted in a significant decrease in steroid production. Conversely, the administration of verapamil (up to 10(-4) M) did not affect steroidogenesis. These results provide evidence that extracellular calcium ions are required for basal production of corticosteroids in amphibians and that Ca2+ influx does not occur through voltage-dependent channels. Since, in the frog, blood Ca2+ concentrations vary in a rather large range, these results suggest that circulating Ca2+ levels may regulate corticosteroid production in these animals.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

Ventricular slices of adult mouse hearts--a new multicellular in vitro model for electrophysiological studies.

AIM: We established a preparation of adult murine ventricular slices suitable for electrophysiological recordings as a new in vitro model of adult myocardium with preserved in vivo tissue structure. METHODS: Short axis slices (thickness: 150 microm) of adult murine ventricles were prepared with a microtome. Sharp glass electrodes were used for measurements of action potentials (APs) at stimulation frequencies of 2 Hz and 10 Hz. Field potential (FP) recordings by means of microelectrode arrays (MEAs) were performed to map excitation spread. RESULTS: APs showed the characteristics of adult murine ventricular APs: (i) a stable resting membrane potential, (ii) a fast upstroke and (iii) a fast phase 1 repolarization. Application of the Na+ channel blocker lidocaine (30 microM) led to a decline of upstroke slope, amplitude and conduction speed. The unspecific K+ channel blocker 4-aminopyridine (5 mM) caused a prolongation of APD50. The excitation spread was homogenous throughout the ventricular wall. CONCLUSION: Adult murine ventricular slices are electrophysiologically intact and respond physiologically to cardioactive drugs. Thus, they provide a new multicellular in vitro model of adult cardiac tissue suitable for electrophysiological investigations, which in future could be used to study the functional integration of stem cells transplanted in infarcted hearts in vivo.     

Differential activation of potassium channels in cerebral and hindquarter arteries of rats during simulated microgravity

The purpose of this study was to test the hypothesis that differential autoregulation of cerebral and hindquarter arteries during simulated microgravity is mediated or modulated by differential activation of K(+) channels in vascular smooth muscle cells (VSMCs) of arteries in different anatomic regions. Sprague-Dawley rats were subjected to 1- and 4-wk tail suspension to simulate the cardiovascular deconditioning effect due to short- and medium-term microgravity. K(+) channel function of VSMCs was studied by pharmacological methods and patch-clamp techniques. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) (K(v)) currents were determined by subtracting the current recorded after applications of 1 mM tetraethylammonium (TEA) and 1 mM TEA + 3 mM 4-aminopyridine (4-AP), respectively, from that of before. For cerebral vessels, the normalized contractility of basilar arterial rings to TEA, a BK(Ca) blocker, and 4-AP, a K(v) blocker, was significantly decreased after 1- and 4-wk simulated microgravity, respectively. VSMCs isolated from the middle cerebral artery branches of suspended rats had a more depolarized membrane potential (E(m)) and a smaller K(+) current density compared with those of control rats. Furthermore, the reduced total current density was due to smaller BK(Ca) and smaller K(v) current density in cerebral VSMCs after 1- and 4-wk tail suspension, respectively. For hindquarter vessels, VSMCs isolated from second- to sixth-order small mesenteric arteries of both 1- and 4-wk suspended rats had a more negative E(m) and larger K(+) current densities for total, BK(Ca), and K(v) currents. These results indicate that differential activation of K(+) channels occur in cerebral and hindquarter VSMCs during short- and medium-term simulated microgravity. It is further suggested that different profiles of channel remodeling might occur in VSMCs as one of the important underlying cellular mechanisms to mediate and modulate differential vascular adaptation during microgravity.     

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.     

The anion transport inhibitor DIDS activates a Ba2+-sensitive K+ flux associated with hepatic exocrine secretion.

4,4'-Diisothiocyanatostilbene-2,2'-disulfonate (DIDS), an anion transport inhibitor and choleretic organic anion, was used to study the relationship between putative DIDS-sensitive K channels and exocrine secretion in the isolated and bile duct cannulated perfused rat liver. Bile flow, DIDS excretion, and effluent perfusate K+ content were measured. DIDS (125 microM) caused a doubling in bile generation concomitant with its appearance in bile, confirming earlier reports. Furthermore, DIDS induced a transient increase in perfusate K+ concentration that peaked prior to the biliary parameters and, after 10 min, reversed to net uptake that fully compensated for the initial release. The K channel blocker Ba2+ (1 mM) strongly inhibited the release phase along with the accompanying choleresis and DIDS excretion. Ouabain (13.5 microM) alone was choleretic and hyperkalemic and, when applied in combination with DIDS, depressed DIDS excretion, choleresis, and DIDS-sensitive K+ uptake. To obtain further evidence for the presence of DIDS-sensitive K channels K+ flux was measured under the influence of different gradients of the cation. Perfusate K+ at 26 and 80 mM changed the DIDS-activated K+ flux from a transient outward to a sustained inward flux, and both DIDS excretion and bile flow decreased. Mean net K+ flux over 20 min DIDS perfusion changed from -1.3 +/-1.1 micromol/g with 5.9 mM K+ to -1304 +/- 55 micromol/g with 80 mM K+ in the perfusate. K+ efflux was fully and reversibly blocked by Ba2+ and influx was ouabain-insensitive, suggesting that the DIDS-activated K+ flux was channel mediated. The results show that a significant fraction of DIDS-induced bile generation is associated with K+ release that may be mediated by Ba(2+)-sensitive K channels, possibly of the inward rectifying type.