Potassium-mediated stimulation of hepatic glycogenolysis

Increased extracellular potassium concentrations ([K+]o) stimulated transient increases in glucose release and 45Ca2+ washout in the perfused rat liver. Stimulated glucose release had a K0.5 of about 26 mM for [K+]o, was not desensitized by successive infusion intervals of increased [K+]o, was not affected by altering the direction of perfusion, was absolutely dependent on the presence of [Ca2+]o, and was blocked by 2 mM cobalt or 10 microM verapamil. The increase in 45Ca2+ washout resulting from increased [K+]o also was blocked by 2 mM cobalt or 10 microM verapamil. Inhibitors of vascular tone (nitroprusside, atriopeptin II), arachidonic acid metabolism (indomethacin, nordihydroguaiaretic acid), and alpha- or beta-adrenergic or muscarinic nerve stimulation/secretion (phentolamine, propranolol, atropine) were unable to inhibit the [K+]o-stimulated glucose release. ATP, ADP, and AMP concentrations in tissue freeze-clamped 2 min after the onset of infusion of 50 mM K+ were not significantly different from control tissue. Glucose release from freshly isolated suspensions or primary cultured monolayers of hepatocytes or from liver slices, all of which responded to glucagon or phenylephrine, did not respond to increased [K+]o. The results indicate that glycogenolysis stimulated by depolarizing gradients of K+ is dependent on an intact perfused vasculature and may be mediated by potential-sensitive Ca2+ channels present in the vascular endothelium of the liver.     

Comparison of changes evoked by GABA (gamma-aminobutyric acid) and anoxia in [K+]o, [Cl-]o, and [Na+]o in stratum pyramidale and stratum radiatum of the guinea pig hippocampus

Ion-selective microelectrode recordings were made to assess a possible contribution of extracellular gamma-aminobutyric acid (GABA) accumulation to early responses evoked in the brain by anoxia and ischemia. Changes evoked by GABA or N2 in [K+]o, [Cl-]o, [Na+]o, and [TMA+]o were recorded in the cell body and dendritic regions of the stratum pyramidale (SP) and stratum radiatum (SR), respectively, of pyramidal neurons in CA1 of guinea pig hippocampal slices. Bath application of GABA (1-10 mM) for approximately 5 min evoked changes in [K+]o and [Cl-]o with respective EC50 levels of 3.8 and 4.1 mM in SP, and 4.7 and 5.6 mM in SR. In SP 5 mM GABA reversibly increased [K+]o and [Cl-]o and decreased [Na+]o; replacement of 95% O2 -5% CO2 by 95% N2 -5% CO2 for a similar period of time evoked changes which were for each ion in the same direction as those with GABA. In SR both GABA and N2 caused increases in [K+]o and decreases in [Cl-]o and [Na+]. The reduction of extracellular space, estimated from levels of [TMA+]o during exposures to GABA and N2, was 5-6% and insufficient to cause the observed changes in ion concentration. Ion changes induced by GABA and N2 were reversibly attenuated by the GABA(A) receptor antagonist bicuculline methiodide (BMI, 100 microM). GABA-evoked changes in [K+]o in SP and SR and [Cl-]o in SP were depressed by > or =90%, and of [Cl-]o in SR by 50%; N2-evoked changes in [K+]o in SP and SR were decreased by 70% and those of [Cl-]o by 50%. BMI blocked delta [Na+]o with both GABA and N2 by 20-30%. It is concluded that during early anoxia: (i) accumulation of GABA and activation of GABA(A) receptors may contribute to the ion changes and play a significant role, and (ii) responses in the dendritic (SR) regions are greater than and (or) differ from those in the somal (SP) layers. A large component of the [K+]o increase may involve a GABA-evoked Ca2+-activated gk, secondary to [Ca2+]i increase. A major part of [Cl-]o changes may arise from GABA-induced g(Cl) and glial efflux, with strong stimulation of active outward transport and anion exchange at SP, and inward Na+/K+/2Cl- co-transport at SR. Na+ influx is attributable mainly to Na+-dependent transmitter uptake, with only a small amount related to GABA(A) receptor activation. Although the release and (or) accumulation of GABA during anoxia might be viewed as potentially protectant, the ultimate role may more likely be an important contribution to toxicity and delayed neuronal death.     

Glycolysis and brain function: [K+]o stimulation of protein synthesis and K+ uptake require glycolysis

Glucose is essentially the sole energy substrate in the normally functioning brain. There are, however, situations in which other substrates can partially substitute for glucose and maintain an apparently normal brain function. However, in no case has it been possible to completely substitute other substrates for glucose and maintain normal brain function. Studies on insulin-induced hypoglycemia suggest that this glucose dependence does not result from its involvement in ATP generation. Two explantation that have been offered are that toxic catabolites arise if nonglucose substrates are oxidized or that glycolysis is necessary to maintain neurotransmitter metabolism. We consider a third basis for the glucose requirement: our past studies have shown that hippocampal slice protein synthesis is activated by small increases in extracellular [K+] ([K+]o), and that this results from activation of K+ uptake into brain cells. We find that this process specifically requires aerobic glycolysis. The basis for the requirement appears to be that [K+]o activation of the Na+-K+ pump is specifically dependent on glycolytically generated energy. Thus, it is possible that glucose is required to maintain normal K+ clearance from the extracellular space during neural activity. This could partially account for the dependence of brain function on glycolysis.     

Adenosine as a signal for ion channel arrest in anoxia-tolerant organisms

Certain freshwater turtles and fish are extremely anoxia-tolerant, capable of surviving hours of anoxia at high temperatures and weeks to months at low temperatures. There is great interest in understanding the cellular mechanisms underlying anoxia-tolerance in these groups because they are anoxia-tolerant vertebrates and because of the far-reaching medical benefits that would be gained. It has become clear that a pre-condition of prolonged anoxic survival must involve the matching of ATP production with ATP utilization to maintain stable ATP levels during anoxia. In most vertebrates, anoxia leads to a severe decrease in ATP production without a concomitant reduction in utilization, which inevitably leads to the catastrophic events associated with cell death or necrosis. Anoxia-tolerant organisms do not increase ATP production when faced with anoxia, but rather decrease utilization to a level that can be met by anaerobic glycolysis alone. Protein synthesis and ion movement across the plasma membrane are the two main targets of regulatory processes that reduce ATP utilization and promote anoxic survival. However, the oxygen sensing and biochemical signaling mechanisms that achieve a coordinated reduction in ATP production and utilization remain unclear. One candidate-signaling compound whose extracellular concentration increases in concert with decreasing oxygen availability is adenosine. Adenosine is known to have profound effects on various aspects of tissue metabolism, including protein synthesis, ion pumping and permeability of ion channels. In this review, I will investigate the role of adenosine in the naturally anoxia-tolerant freshwater turtle and goldfish and give an overview of pathways by which adenosine concentrations are regulated.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Effect of Extracellular Potassium on Ouabain-Sensitive Consumption of High-Energy Phosphate by Crayfish Giant Axons: A Study of the Energy Requirement for Transport in the Steady State

Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport "coupling ratio" and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady-state consumption of high-energy phosphate (approximately P) [ATP and phospho-L-arginine (Arg-P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total approximately P consumption (Q approximately P) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q approximately P averaged 52.8 +/- 4.7%/h (n = 44) of the initial [ATP] + [Arg-P]. Unlike total Q approximately P, the ouabain-sensitive portion of Q approximately P was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q approximately P to 8%/h, a result indicating that 85% of the total Q approximately P was ouabain sensitive. For 1.35 mM K+o, the ouabain-sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mM K+o, 41%. There was a small but significant increase in the ouabain-sensitive Q approximately P at 21.6 mM K+o, compared with Q approximately P at 5.4 mM K+o. The pattern of effect of [K+]o on Q approximately P was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/approximately P stoichiometry of 3, an actual ratio of JNa/approximately P stoichiometry of approximately 33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero-membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non-steady-state conditions.     

Anoxia-induced changes in reactive oxygen species and cyclic nucleotides in the painted turtle

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduction of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxygen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle’s metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20–22°C. Turtle brain ROS production decreased 25% within 10 min of cyanide or N₂-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of complex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner suggestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

Glucose-induced oscillatory changes in extracellular ionized potassium concentration in mouse islets of Langerhans.

Liquid membrane [K+]-sensitive microelectrodes (1-2 micron tip diameter) were used to measure the extracellular ionized potassium concentration in mouse pancreatic islets of Langerhans. With the tip of the microelectrode at the surface of the islet, the time course of the [K+]-sensitive electrode potential changes in response to the application of rapid changes in [K+]o (from 1.25 to 5 mM), could be reproduced by the equation for K+-diffusion through a 100-micron-thick unstirred layer around the islet (diffusion coefficient for K+ at 27 degrees C, DK,o, taken as 1.83 X 10(-5) cm2/s). The time to reach 63% of the steady-state electrode response with the tip in the chamber at the surface of the islet was from 5 to 6 s. When the tip of the [K+]-sensitive electrode was placed in the islet tissue, the time for the response to reach 63% of the steady-state level increased. The time course of the [K+]-sensitive electrode response could be reproduced using the same diffusion model assuming that K+ diffusion into the islet tissue takes place in a tortuous intercellular path with an apparent diffusion coefficient, DK,I, about half of DK,o, in series with the unstirred layer around the islet. In the absence of glucose the potassium concentration in the extracellular space, [K+]I, was found to be higher than the concentration in the external modified Krebs solution, [K+]o. The difference in concentration [K+]I - [K+]o was greater when [K+]o was smaller than 2 mM. In the presence of glucose (between 11 and 16 mM), under steady-state conditions, small oscillatory changes in the [K+], (1.48 +/- 0.94 mM) were detected. Simultaneous recording of membrane potential from one B-cell and [K+], in the same islet indicated that the potassium concentration increased during the active phase of the bursts of electrical activity. Maximum concentration in the intercellular was reached near the end of the active phase of the bursts. We propose that the space between islet cells constitutes a restricted diffusion system where potassium accumulates during the transient activation of potassium channels.     

Determinants of Deoxyglucose Uptake in Cultured Astrocytes: The Role of the Sodium Pump

Glucose utilization in primary cell cultures of mouse cerebral astrocytes was studied by measuring uptake of tracer concentrations of [3H]2-deoxyglucose ([3H]2-DG). The resting rate of glucose utilization, estimated at an extracellular K+ concentration ([K+]o) of 5.4 mM, was high (7.5 nmol glucose/mg protein/min) and was similar in morphologically undifferentiated and "differentiated" (dibutyryl cyclic AMP-pretreated) cultures. Resting uptake of [3H]2-DG was depressed by ouabain, by reducing [K+]o, and by cooling. These observations suggest that resting glucose utilization in astrocytes was dependent on sodium pump activity. Sodium pump-dependent uptake in 2-3-week-old cultures was about 50% of total [3H]2-DG uptake but this fraction declined with culture age from 1 to 5 weeks. Uptake was not affected by changes in extracellular bicarbonate concentration ([HCO3-]o) in the range of 5-50 mM but was significantly reduced in bicarbonate-free solution. At high [HCO3-]o (50 mM) uptake was insensitive to pH (pH 6-8), whereas at low [HCO3-]o (less than 5 mM) uptake was markedly pH-dependent. Elevation of [K+]o from 2.3 mM to 14.2-20 mM (corresponding to extremes of the physiological range of [K+]o) resulted in a 35-43% increase in [3H]2-DG uptake that was not affected by culture age or by morphological differentiation. Our results indicate a high apparent rate of glucose utilization in astrocytes. This rate is dynamically responsive to changes in extracellular K+ concentration in the physiological range and is partially dependent on sodium pump activity.     

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.     

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.     

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.     

Thyroid status and beta-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization.

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.     

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.     

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic beta-cells during blockade of K(ATP) channels.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.