Electrical bursting in islet beta cells.
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Animals↗
Biomedical subjects
D L Cook
Publications and source records attributed to D L Cook.
Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic beta-cells and HIT cells.
ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic beta-cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 microM and as high as 500 microM, with maximal activation between 10 and 100 microM ADP in mouse beta-cell membrane patches. At concentrations greater than 500 microM, ADP inhibited K(ATP) channels while 10 mM virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mM ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of beta-cell membrane potential, increasing ADP in the 10 and 100 microM range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize beta-cells. The results suggest that (i) K(ATP)-channel activity is not well predicted by the "ATP/ADP ratio," and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10-100 microM range as suggested by biochemical studies.
Adenosine Diphosphate↗
Cocaine-induced rhabdomyolysis. Findings on bone scintigraphy.
During the examination of a young patient with acute renal failure, bone imaging was performed for evaluation of possible osteomyelitis. Marked soft tissue activity was seen on the bone scan. The patient admitted to recent intravenous cocaine use. Vasoconstriction from cocaine use was thought to induce rhabdomyolysis, which led to acute renal failure with myoglobulinuria.
Acute Kidney Injury↗
Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells.
Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidly-inactivating component had a time constant of 75 +/- 5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inactivating component had a time constant of 2750 +/- 280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between -100 and -40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 microM) when a holding potential of -100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1-10 microM) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.
Activation Analysis↗
Specificity of tetraethylammonium and quinine for three K channels in insulin-secreting cells.
The effects of tetraethylammonium (TEA) and quinine on Ca-activated [K(Ca)], ATP-sensitive [K(ATP)]K channels and delayed-rectifier K current [K(dr)] have been studied in cultured insulin-secreting HIT cells using the patch-clamp technique. K(Ca) and K(ATP) channels were identified in excised, outside/out patches using physiological solutions and had unitary conductances of 60.8 +/- 1.3 pS (n = 31) and 15.4 +/- 0.3 pS (n = 40), respectively. Macroscopic K(dr) current (peak current = 607 +/- 100 pA at +50 mV, n = 14) were recorded in the presence of 100 microM cadmium and 0.5 microM tetrodotoxin. Tetraethylammonium (TEA) blocked all three channel types but was more effective on K(Ca) channels (EC50 = 0.15 mM) than on K(ATP) channels (EC50 = 15 mM) or K(dr) currents (EC50 = 3 mM). Quinine also blocked all three currents but was less effective on K(Ca) channels (EC50 = 0.3 mM) while equally effective against K(ATP) channels and K(dr) currents (EC50 = 0.025 mM). TEA blocked K(Ca) and K(ATP) channels by reducing their single-channel conductances and decreasing the probability of K(ATP) channel opening. Quinine blocked K(Ca) channels by reducing the single-channel conductance, but blocked K(ATP) channels by reducing the probability of channel opening. Reinterpretation of previous microelectrode studies in light of these findings suggest that, (i) only K(ATP) channels are active in low glucose, (ii) both K(Ca) and K(dr) channels may assist Ca-spike repolarization, and (iii) K(Ca) channels play no role in forming the burst pattern of Ca spiking in the B cell.
Adenosine Triphosphate↗
Pancreatic B cells are bursting, but how?
Insulin secretogogues have long been known to stimulate and modulate bursting electrical activity in pancreatic islet B cells and thereby supply extracellular Ca2+ for the exocytosis of insulin. Recent results have ruled out a long-held hypothesis for the mechanism of burst formation that postulated key roles for intracellular Ca2+ accumulation and activation of Ca(2+)-activated K+ channels. Here, we present an alternative hypotheses based on a persistent Ca2+ conductance and, possibly, phasic activation of ATP-sensitive K+ channels. These hypotheses are compared with mechanisms of bursting proposed for invertebrate and mammalian neurons.
Animals↗
Secretory patterns of LH and FSH during development and hypothalamic and hypophysial characteristics following development of steroid-induced ovarian follicular cysts in dairy cattle.
Two experiments were conducted to (1) investigate developmental endocrinology of ovarian follicular cysts (cysts) in cattle and (2) evaluate effects of cysts on hypothalamic and hypophysial characteristics. Cysts were induced with oestradiol-17 beta (15 mg) and progesterone (37.5 mg) dissolved in alcohol and injected s.c. twice daily for 7 days. Cysts were defined as the presence of follicular structures (which may or may not have been the same structure) of 2.0 cm in diameter or greater that were present for 10 days without ovulation and corpus luteum development. In Exp. 1,22 non-lactating, non-pregnant Holstein cows were allocated to 3 groups. Beginning on Day 5 (oestrus = Day 0) of the oestrous cycle, 7 cows (Controls) were treated with twice daily s.c. injections of ethanol (2 ml/injection) for 7 days. Luteolysis was then induced with PGF-2 alpha and blood samples were collected daily every 15 min for 6 h from the morning after the PGF-2 alpha injection (Day 13) until oestrus. Steroids to induce cysts were injected as previously described into the remaining cows (N = 15). Three blood samples were collected at 15-min intervals every 12 h throughout the experimental period. Additional blood samples were collected every 15 min for 6 h on a twice weekly basis. After steroid injections, follicular and luteal structures on ovaries were not detected via rectal palpation for a period of 36 +/- 4 days (static phase). Then follicles developed which ovulated within 3-7 days (non-cystic; N = 7) or increased in size with follicular structures present for 10 days (cystic; N = 8). Mean (+/- s.e.m.) concentrations of LH, FSH, oestradiol-17 beta and progesterone in serum remained low and were not different during the static phase between cows that subsequently developed cysts or ovulated. During the follicular phase, mean serum concentration of LH (ng/ml) was higher (P less than 0.1) in cows with cysts (2.9 +/- 0.2) than in cows without cysts (1.1 +/- 0.1) or control cows (1.4 +/- 0.2). In addition, LH pulse frequency (pulses/6 h) and amplitude (ng/ml) were higher (P less than 0.1) in cows with cysts (3.6 +/- 0.3 and 2.2 +/- 0.3, respectively) than in non-cystic (2.3 +/- 0.2 and 1.0 +/- 0.2, respectively) and control (1.8 +/- 0.1 and 1.1 +/- 0.2, respectively) groups during the follicular phase. There were no differences in the FSH, oestradiol-17 beta or progesterone characteristics in cows of any of the 3 groups during the follicular phase.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The oral hypoglycemic agent, U-56324, inhibits the activity of ATP-sensitive potassium channels in cell-free membrane patches from cultured mouse pancreatic B-cells.
U-56324, a hypoglycemic agent derived from nicotinic acid, inhibited the activity of ATP-sensitive potassium channels in excised patches from mouse pancreatic B-cells. The effect of U-56324 on channel activity was reversible and concentration-dependent while it had no effect on single channel conductance. The positional isomer, U-59588, which has relatively little hypoglycemic activity, had no effect on channel properties. U-56324, at the same concentrations, had no effect on calcium-activated potassium channels. The basis for the potentially antidiabetic properties of U-56324 may therefore be due to direct and specific inhibition of ATP-sensitive potassium channels.
Adenosine Triphosphate↗
The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic beta-cells.
In recent years, the electrical burst activity of the insulin releasing pancreatic beta-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K+ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca2+ level in the pancreatic beta-cell.
Adenosine Diphosphate↗
Fate and turnover rate of ovarian follicular cysts in dairy cattle.
Non-lactating, multiparous dairy cows diagnosed as having cysts by palpation per rectum were used. Cysts were induced with oestradiol-17 beta (15 mg) and progesterone (37.5 mg) dissolved in ethanol and injected s.c. twice daily for 7 days. Following initial diagnosis of cysts, ovaries were exposed by midventral laparotomy, and the perimeter of the base of each cyst was marked with subepithelial injections of charcoal. Ovaries were removed from cows by transvaginal incision at 10 days (Group 1; N = 8), 20 days (Group 2; N = 8), or 40 days (Group 3; N = 7) after marking of cysts. Ovaries were examined for structures present and their relationship to the marked site. Corpora lutea with ovulation papilla were present in 7/23 cows (1/8, 4/8 and 2/7, for Groups 1, 2 and 3, respectively). In these 7 cows, corpora lutea were at a site different from the original structure that was marked. Marked structures persisted for the duration of the experimental period in 1 and 2 cows, in Groups 1 and 3, respectively. In the remaining 13 cows, new large follicular structures (cysts) were present at a site other than that marked with charcoal. These structures developed on the ovary contralateral to the one originally marked in 9 of 13 cows. Cysts are therefore dynamic in nature and may persist or may be replaced by others.
Animals↗
Calcium current inactivation in insulin-secreting cells is mediated by calcium influx and membrane depolarization.
Inactivation of voltage-dependent calcium currents was studied in single, dissociated insulin-secreting HIT cells voltage-clamped by the whole-cell patch-clamp method at room temperature. Na and K currents were suppressed by tetrodotoxin, tetraethylammonium, ATP, 4-aminopyridine and Cs. Ca currents activated in less than 10 ms by depolarizations beyond -50 mV from a holding potential of -100 mV and were identified, as in previous studies, by their sensitivity to divalent cation blockade and permeability to Ba as a charge carrier. Sustained depolarization revealed two kinetically distinct phases of inactivation: a rapid phase inactivated approximately 50% of the current in less than 100 ms while the remaining current was inactivated over the next 10-20 s. Rapid inactivation appeared to be due to Ca2+ influx since it was slowed markedly when Ba2+ was used as the current carrier, while the degree of inactivation increased and decreased with increasing depolarization in direct parallel with the U-shaped current-voltage relationship for inward Ca current. Slow inactivation appeared to be voltage-dependent since current could be inactivated (by approximately 20%) by 10 s long depolarizations to potentials below the threshold for activating Ca current, slow time constants of inactivation were voltage-dependent and slow inactivation persisted when Ca was replaced with Ba. Ca currents with low activation thresholds (in the -50 to -30 mV range) appeared to be preferentially inactivated by the rapid Ca-dependent mechanism. Recovery of slowly inactivated Ca current was very slow and currents inactivated by larger depolarizations required longer recovery time than those elicited by smaller depolarizations. Rapid and slow inactivation mechanisms may be important in understanding the fast spiking and slow plateau depolarizations seen in pancreatic B-cells exposed to stimulatory levels of glucose.
Animals↗
Expression of a rapid, low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering.
Depolarization-activated outward currents ranging in amplitude from 100-1000 pA were studied in cultured, insulin-secreting HIT cells and mouse B-cells using the whole-cell patch clamp. Outward current was identified as a K current since it was blocked by K channel blockers and its tail current reversed near EK. The K currents of HIT cells dialyzed with internal solutions containing 0.1-10 mM EGTA with no added calcium (Ca), or 10 mM EGTA with 2 mM added Ca, activated rapidly with depolarization. However, the stronger Ca buffer BAPTA (5 mM; no added Ca) blocked the rapidly activating current to reveal an underlying more slowly activating K current. With intracellular EGTA, application of the Ca channel blocker cadmium mimicked the effect of intracellular BAPTA. These data suggest that the rapid K current was mediated by low-voltage threshold, Ca-activated K channels while the slower K current was mediated by high threshold delayed rectifier K channels. Mouse B-cells also had both K current components. Dialyzing these cells with either BAPTA (5 mM, no added Ca) or high EGTA (10 mM with 2 mM Ca) blocked the rapid Ca-activated K current observed when cells were filled with 0.1 to 1 mM EGTA. It is concluded that the extent of Ca-activated K current activation in either HIT or adult mouse B-cells depends on the degree of intracellular Ca buffering.
Animals↗
Tolbutamide as mimic of glucose on beta-cell electrical activity. ATP-sensitive K+ channels as common pathway for both stimuli.
It is accepted for insulin-secreting cells in culture that the closure of ATP-sensitive K+ channels causes the glucose-dependent depolarization of pancreatic beta-cells seen at subthreshold levels (less than 100 mg/dl) of glucose. The question remains for the more thoroughly studied beta-cells in freshly dissected intact islets, however, whether closure of these channels is responsible for subthreshold glucose-dependent depolarization and suprathreshold glucose-dependent regulation of membrane electrical activity. To answer this, we took advantage of the ability of tolbutamide, an orally active antidiabetic agent, to specifically inhibit ATP-sensitive K+ channels in pancreatic beta-cells to determine whether these channels are active at sub- and suprathreshold levels of glucose and whether channel closure by tolbutamide reproduces the electrophysiological effects of glucose stimulation. We recorded membrane electrical activity from freshly dissected adult mouse pancreatic islets exposed to various levels of glucose and tolbutamide. As previously found by others, tolbutamide depolarizes islet cells in the absence of glucose, but we have found that, although the depolarization can trigger Ca2+ action potentials (spikes), a glucose-dependent permissive factor may be required for the normal bursting pattern of spiking. More significantly, we found that, unlike other beta-cell stimuli, tolbutamide specifically mimics the effects of glucose stimulation on the pattern of suprathreshold electrical activity. The effects were seen with levels of tolbutamide that correspond to those required to inhibit ATP-sensitive K+ channels. These data suggest that ATP-sensitive K+ channels are active at sub- and suprathreshold levels of glucose and may be the sole pathway by which either glucose or tolbutamide depolarizes beta-cells and controls beta-cell electrical activity.
Action Potentials↗
Influence of dexamethasone, progesterone, gonadotropin-releasing hormone, and testosterone on estrous behavior of estradiol-treated ovariectomized heifers.
In Experiment 1, 12 ovariectomized heifers were assigned to receive weekly hormone treatments in a replicated 6 x 6 Latin square design. Hormonal treatments were given as two simultaneous injections i.m. and consisted of: 1) 2 ml propylene glycol and 2 ml propylene glycol; 2) .5 mg estradiol benzoate and 2 ml propylene glycol; 3) .5 mg estradiol benzoate and 4 mg dexamethasone; 4) .5 mg estradiol benzoate and 10 mg progesterone; 5) .5 mg estradiol benzoate and .4 mg GnRH; and 6) .5 mg estradiol benzoate and 12.5 mg testosterone propionate. The .5 mg estradiol benzoate and 4 mg dexamethasone treatment reduced the percentage of heifers in estrus compared with the .5 mg estradiol benzoate and 2 ml propylene glycol treatment. In Experiment 2, 16 ovariectomized heifers were used in four replicates of a 4 x 4 Latin square design to determine if pretreatment with progesterone potentiated the actions of estradiol. Hormonal treatments i.m. consisted of: 1) 0 mg progesterone and .2 mg estradiol benzoate; 2) 50 mg progesterone and .2 mg estradiol benzoate; 3) 0 mg progesterone and .5 mg estradiol benzoate; and 4) 50 mg progesterone and .5 mg estradiol benzoate. Progesterone pretreatment, at either dosage of estradiol benzoate, did not increase the percentage of heifers in estrus. Based on these observations, we conclude that: 1) dexamethasone inhibited estrus in estradiol-treated ovariectomized heifers and 2) progesterone pretreatment did not potentiate the actions of estradiol in ovariectomized heifers.
Animals↗
Evidence for two calcium currents in insulin-secreting cells.
Inward currents were measured in cultured rat pancreatic islet cells using the whole-cell patch-clamp technique. Depolarization elicited both transient and more sustained inward currents. The transient current was blocked by external Na removal, TTX or depolarizing holding potentials while the sustained current was blocked by external Ca removal or the addition of Co, Ni or Cd ions to the external solution. Replacing Ca with Ba increased the amplitude of the sustained current and decreased its rate of decay. These results suggest that the transient current was due to the activation of Na channels while the sustained current was mediated by Ca channels. Two characteristics of the data suggested that two Ca currents may contribute to the sustained current. First, a second shoulder, or hump, was observed on the descending limb of the Ca current I-V of many cells, suggesting that they may possess two Ca channels that activate at different voltages. Second, the low and high voltage components of the I-V were differentially suppressed by Cd. Neither of the Ca current components was very sensitive to the Ca channel agonist BAY K 8644. These results were confirmed in HIT cells, another insulin-secreting preparation.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
ATP-sensitive K+ channels in pancreatic beta-cells. Spare-channel hypothesis.
Since their discovery in pancreatic beta-cells, ATP-sensitive K+ channels in the cell membrane have been thought to mediate glucose-induced beta-cell depolarization, which is required for triggering the voltage-dependent Ca2+ uptake subserving insulin release. The theory is that metabolism of glucose (and other fuel molecules) increases intracellular ATP or possibly other metabolites that diffuse to the membrane and inhibit the opening of ATP-sensitive K+ channels. This slows the efflux of positively charged K+ and depolarizes the cell. A recurrent source of confusion regarding this idea stems from the early observation that these channels are so exquisitely sensitive to intracellular ATP that channel opening is predicted to be approximately 99% inhibited under physiological conditions. To account for this apparent discrepancy, various mechanisms have been proposed that might render the channels less sensitive to intracellular ATP. We use a simple mathematical model to demonstrate that there is no major discrepancy and that, in fact, given the electrophysiological mechanisms existing in the beta-cell, the extreme sensitivity of the channels to ATP is appropriate and even mandatory for their physiological function.
Adenosine Triphosphate↗
Voltage-gated Ca current in pancreatic islet beta-cells.
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Animals↗
Glucose suppresses ATP-inhibited K-channels in pancreatic beta-cells.
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Adenosine Triphosphate↗