Inosine partially mimics the effects of glucose on ionic fluxes,electrical activity,and insulin release in mouse pancreatic B-cells

The purine ribonucleoside inosine is known to be metabolized in islet cells (its ribose moiety feeds into the pentose-phosphate cycle) and stimulate insulin release, but the mechanisms of this stimulation have not been established. These were investigated with mouse islets. In the absence of glucose, 5 mM inosine decreased⁸⁶Rb⁺ efflux from islet cells, depolarized the B-cell membrane, induced electrical activity (slow waves of membrane potential with bursts of spikes on the plateau), accelarated⁴⁵Ca²⁺ efflux and stimulated insulin release with the same efficiency as 10 mM glucose. Raising the concentration of inosine to 20 mM only had a slight further effect and, in particular, failed to cause persistent depolarization of the B-cell membrane. The electrical activity triggered by inosine was blocked by cobalt, and the stimulation of⁴⁵Ca²⁺ efflux and insulin release was abolished in a Ca²⁺-free medium. The effects of 10 mM glucose on electrical activity,⁴⁵Ca²⁺ efflux and insulin release were augmented by as little as 0.5 mM inosine. All effects of inosine were abolished by an inhibitor of nucleoside transport (nitrobenzylthioguanosine) and markedly impaired by inhibitors of nucleoside phosphorylase (formycin B) or of glycolysis (iodoacetate). In conclusion, inosine metabolism in B-cells induces insulin release by triggering the same sequence of events as glucose metabolism: a decrease of K⁺ permeability of the B-cell membrane, leading to depolarization and activation of voltage-dependent Ca channels.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2

RORSMAN P. & ABRAHAMSSON, H. 1985. Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca²⁺. Acta Physiol Scand 125 , 639–647. Received 18 March 1985, accepted 28 May 1985. ISSN 0001–6772. Department of Medical Cell Biology, Biomedicum, University of Uppsala, Sweden. The effects of various stimulants of insulin release on cytosolic free Ca²⁺, [Ca²⁺]i, in dispersed and cultured pancreatic β-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca²⁺]_i was 158 ± 7 nM. After increasing glucose to 20 mM there was a lag-period of 1–2 min before [Ca²⁺]_i gradually rose, reaching a new plateau 60% higher after 5–6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca²⁺]_i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the β-cell function was evident from a marked increase of cytosolic [Ca²⁺]_i after depolarization evoked by high extracellular K⁺. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca²⁺]_i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca²⁺]_i rather than by increasing the cytosolic concentration of the ion.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Role of voltage- and Ca2+-dependent K+ channels in the control of glucose-induced electrical activity in pancreatic B-cells

Low concentrations of tetraethylammonium chloride (TEA), which inhibit voltage- and Ca²⁺-sensitive K⁺ channels (K⁺-VCa channels), were used to investigate whether these channels play a role in the control of glucose-induced electrical activity (slow waves with spikes) in mouse pancreatic B-cells. Addition of 2 mM TEA to a medium containing 0, 3 or 6 mM glucose had no effect on the membrane potential of B-cells or on ⁸⁶Rb⁺ efflux and insulin release from isolated islets. In 10 mM glucose, 0.5–2 mM TEA produced a concentration-dependent increase in spike amplitude without modifying slow-wave duration or frequency. Insulin release was only slightly increased under these conditions. In conclusion, K⁺-VCa channels are not operative when the B-cell membrane is not depolarized (in low glucose). They appear to play a role in the repolarization of the spikes but not in that of the slow waves. In contrast to ATP-sensitive K⁺ channels, K⁺-VCa channels are not a target on which glucose acts to regulate electrical activity in B-cells and, hence, insulin release.     

Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans

Sodium azide (NaN₃), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10–15 min) application followed by removal of 3 mM NaN₃ results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3–5 mM). In addition, application of NaN₃, even at very low [Ca²⁺]_o, reversibly increases cytosolic Ca²⁺ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca²⁺, by itself, is not sufficient to trigger insulin secretion.Deceased     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Dissociation of K+-induced tension and cellular Ca2+ retention in vascular and intestinal smooth muscle in normoxia and hypoxia

In experiments on smooth muscle preparations of rabbit aorta and guinea pig taenia coli, replacement of the external Na⁺ with K⁺ produced sustained contraction. When external K⁺ concentration was increased, cellular Ca²⁺ retention as measured by a modified lanthanum technique increased. However, when K⁺ concentration was above 80 mM, the tension decreased despite an increase in Ca²⁺ retention. Maximum amount of Ca²⁺ retained was 1280 nmol/g in aorta and 980 nmol/g in taenia coli while the control values for both tissues were approximately 430 nmol/g when the external Ca²⁺ concentration was 2.5 mM. Under hypoxia (N₂ aeration), sustained contraction was induced by 80 mM K⁺ in aorta and by 45.4 mM K⁺ (and 55 mM glucose) in taenia coli. However, no increase in the cellular Ca²⁺ retention was observed under these conditions. During the K⁺-induced sustained contraction in aorta, introduction of N₂ transiently increased, while readmission of O₂ transiently decreased the muscle tension. In taenia coli, the introduction of N₂ decreased the sustained contractile tension probably because of an ATP deficiency, while the readmission of O₂ further decreased the tension trasniently. From these results, it is concluded that, in the presence of a high concentration of K⁺, external Ca²⁺ enters the cell and activates the contractile machinery. A part of the cellular Ca²⁺ is taken up by mitochondria under normoxic but not under hypoxic conditions.     

Different effects of depolarization and muscarinic stimulation on the Ca2+/force relationship during the contraction-relaxation cycle in the guinea pig ileum

The effects of K⁺ depolarization and of the muscarinic agonist carbachol on [Ca²⁺]_i and force were investigated in smooth muscle sheets of the longitudinal layer of the ileum loaded with Fura-2. K⁺ -rich solutions increased [Ca²⁺]_i and force to an initial peak value, which was determined by the concentration of [K⁺]_o. Thereafter, [Ca²⁺]_i and force declined to a lower maintained level. The Ca²⁺/force relationship observed during this contraction-relaxation cycle is represented by a clockwise hysteresis loop. At 140 mM [K⁺]_o, this loop consisted of three components while at lower [K⁺]_o a two-component loop was observed. The stimulation with 0.1 mM carbachol resulted in a transient increase of [Ca²⁺]_i and force followed by a continuous decline of these parameters despite the presence of the drug. Its EC₅₀ of relaxation was around 270 nM [Ca²⁺]_i. The Ca²⁺/force relationship proceeded along a counterclockwise hysteresis loop during the contraction-relaxation cycle. The extent of this loop decreased but remained unaltered in its direction during repeated stimulation with carbachol. These results suggest that (a) both agonists increase force and [Ca²⁺]_i during stimulation; (b) during depolarization with K⁺, desensitization to Ca²⁺ occurs resulting in a clockwise hysteresis loop; (c) during carbachol stimulation, a counterclockwise hysteresis is observed. This could be due to an increased sensitivity to Ca²⁺ mainly in tonic smooth muscle. These observations might be explained by a modulation of the Ca²⁺ sensitivity by sensitizing and desensitizing mechanisms. These modulations during different stimuli could be due to different myosin light-chain kinase/myosin light-chain phosphatase ratios.     

Regulatory volume increase after secretory volume decrease in colonic epithelial cells under muscarinic stimulation

To address the question of whether colonic secretory cells change their volume in response to carbachol (CCh) stimulation and, if so, the mechanisms involved therein, we used two-photon laser scanning microscopy to measure the volume of individual epithelial cells in the fundus region of crypts isolated from the guinea-pig distal colon. We also measured the volume of human colonic epithelial T84 cells using an electronic sizing technique. Both types of colonocytes responded to stimulation by CCh with shrinkage and then underwent a regulatory volume increase (RVI), even during continued stimulation by CCh. The secretory volume decrease (SVD) induced by CCh was antagonized by atropine, BAPTA loading and niflumic acid, a blocker of Ca²⁺-activated Cl^– channels. An increase in the intracellular free [Ca²⁺] was observed with fura-2 during these volume responses to CCh. Removal of all Na⁺ or K⁺ or of most of the Cl^– from the extracellular solution abolished the RVI, but not the preceding SVD. The RVI, but not the preceding SVD, was abolished by bumetanide, a blocker of the Na⁺-K⁺-2Cl^– cotransporter. We conclude that guinea-pig crypt colonocytes and human T84 cells exhibit a cytosolic Ca²⁺-dependent SVD and undergo a subsequent RVI that is dependent on the operation of Na⁺-K⁺-2Cl^– cotransporters.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic β-cells

The influence of the antidiabetic sulphonylurea tolbutamide on K⁺ channels of mouse pancreatic -cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K⁺ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of -cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 M. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K⁺ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca²⁺-dependent K⁺ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K⁺ channels.     

Effects of urea on K+ fluxes and cell volume in perfused rat liver

Exposure of the perfused rat liver to a perfusate made hyperosmotic by the presence of 200 mmol l^–1glucose led, as expected, to marked, transient hepatocellular shrinkage followed by volume-regulatory net K⁺ uptake. However, even after this volume-regulatory K⁺ uptake had ceased, the liver cells are still slightly shrunken. Withdrawal of glucose from the perfusate resulted in marked transient cell swelling, net K⁺ release from the liver and restoration of cell volume. However, when the Krebs-Henseleit perfusate was made hyperosmotic by the presence of urea (20–300 mM), there was no immediate decrease in liver mass, yet a slight and persistent cell shrinkage developing 2 min after the onset of exposure to urea. Surprisingly, urea induced concentration-dependent net K⁺ efflux from the liver and removal of urea net K⁺ reuptake from the inflowing perfusate. The urea (200 mM)-induced net K⁺ release resembled that observed following a lowering of the influent [NaCl]: making the perfusate hypoosmotic (245 mosmol l^–1, by reducing influent [NaCl] by 30 mM) gave roughly the same K⁺ response as hyperosmotic exposure (505 mosmol/l) resulting from the presence of 200 mM urea. The urea-induced K⁺ efflux was not inhibited in the presence of ouabain (1 mM), or in Ca⁺⁺-free perfusion, but was modified in the presence of quinidine (1 mM) or Ba⁺⁺ (1 mM). The direction in which the liver was perfused had no effect on the urea-induced net K⁺ release. Electrophysiological studies showed that urea led to quinidine-sensitive hyperpolarization and increase in K⁺ selectivity of plasma membranes, suggesting opening of K⁺ channels in the hepatocyte plasma membrane in response to urea. The data suggest that urea, but not glucose, enters the hepatocyte as quickly as water. Furthermore, urea at high concentrations apparently leads to K⁺ efflux from the hepatocyte and cell shrinkage, possibly due to opening of K⁺ channels in the hepatocyte plasma membrane.     

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based perforated patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca²⁺]_in resulting from Ca²⁺ release from caffeine-ryanodine-sensitive internal stores. In addition to Ca²⁺ release, IBMX produced a biphasic membrane current response comprised of an inward current transiently interrupted by outward current. The onset of the inward current slightly preceded the onset of the [Ca²⁺]_in transient, while the interrupting outward current developed synchronously with the [Ca²⁺]_in rise. The development of IBMX-induced outward current ultimately needed the [Ca²⁺]_in elevation. After the depletion of Ca²⁺ stores by IBMX or caffeine exposure, the subsequent IBMX challenge failed to produce both the [Ca²⁺]_in transient and outward membrane current, although the inward current remained unchanged. Both components of the IBMX-induced membrane current response had a reversal potential close to the K⁺ equilibrium potential and the IBMX-induced membrane current response disappeared while dialysing the cell interior with K⁺-free, Cs⁺-containing solutions suggesting their association with K⁺ channel activity. External administration of 10 mM tetraethylammonium chloride (TEA-Cl) evoked an inward current similar to that observed in response to IBMX; in the presence of TEA-Cl, IBMX application was almost unable to induce additional inward current. IBMX (5 mM) effectively (50%) inhibited K⁺ currents evoked by step depolarizations of membrane potential. We suggest that IBMX affects membrane permeability via activation of Ca²⁺-regulated K⁺ channels and direct inhibition of TEA-sensitive K⁺ channels.     

Paradoxical decrease in cytosolic calcium with increasing depolarization by potassium in guinea-pig mesotubarium smooth muscle

The free intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. [Ca²⁺]_i and force were maximal at a relatively low (30 mM) concentration of extracellular K⁺ ([K⁺]_o), and declined at 90 and 140 mM K⁺. Plateau values of both [Ca²⁺]_i and force were higher in the presence of 5 · 10^–6 M ryanodine, indicating that the sarcoplasmic reticulum (SR) contributes to the decline with depolarization. Force and [Ca²⁺]_i at 90 mM K⁺ were both lower then the high-K⁺ solution was applied after a period in 30 mM K⁺ than after a period in normal solution (5.9 mM K⁺), consistent with inactivation of Ca²⁺ channels during prolonged depolarization. Addition of carbachol to the depolarized muscle caused a maintained increase in force without maintained increase in [Ca²⁺]_i. We conclude that the decrease in force at increased [K⁺]_o (the calcium-potassium paradox) is due to a membrane-potential-mediated decrease in [Ca²⁺]_i and, to a lesser extent, to desensitization of the contractile-regulatory apparatus to Ca²⁺.     

The calcium channel antagonist diltiazem effectively blocks two types of potassium channels in the neuronal membranes

Effect of the Ca²⁺-channel antagonist diltiazem on potential-operated Ca²⁺ and K⁺ currents was studied on isolated edible snail neurons by a two-microelectrode patch-clamp technique. Diltiazem in a concentration of 0.1 mM inhibits Ca²⁺ current, high-threshold Ca²⁺-dependent K⁺ current, and Ca²⁺-independent K⁺ current and has no effect on low-threshold K⁺ current and leakage current. It is suggested that therapeutic effect of diltiazem is mediated through blockade of Ca²⁺ and K⁺ channels. Tranlated fromByulleten' Eksperimental'noi biologii i Meditsiny, Vol. 124, No. 9, pp. 271–274. September, 1997     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.