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Biomedical subjects

A E Boyd

Publications and source records attributed to A E Boyd.

At least 19 recordsLinked to original sources
PubMed1992

Octreotide treatment of acromegaly. A randomized, multicenter study.

OBJECTIVE: To determine the effects of the somatostatin analog, octreotide acetate, in patients with acromegaly. DESIGN: Double-blind, randomized trial. SETTING: Fourteen university-affiliated medical centers. PATIENTS: One hundred fifteen acromegalic patients, 70% of whom had persistent disease after pituitary surgery or radiotherapy. INTERVENTION: Subcutaneous octreotide, 100 micrograms, or placebo every 8 hours for 4 weeks. Four weeks after the end of treatment, patients were randomized to receive 100 or 250 micrograms octreotide subcutaneously every 8 hours for 6 months. RESULTS: After 2 weeks of treatment, a single 100-micrograms injection reduced mean serum growth hormone (GH) to 30% of the pretreatment concentration within 2 hours. The integrated mean GH level was reduced over 8 hours from 39 +/- 11 micrograms/L to 9 +/- 2 micrograms/L (P less than 0.001). Mean plasma insulin-like growth factor-1 (IGF-1) was reduced from 5100 +/- 400 U/L to 2400 +/- 400 U/L (P less than 0.001). After 6 months, the mean GH was reduced from 39 +/- 13 to 15 +/- 4 micrograms/L by 300 micrograms of octreotide and from 29 +/- 5 micrograms/L to 9 +/- 2 micrograms/L by 750 micrograms of octreotide daily. The mean IGF-1 concentration was suppressed to 2100 +/- 300 and 2500 +/- 400 U/L after 300 and 750 micrograms octreotide, respectively. Integrated mean GH levels were reduced to < 5 micrograms/L in 53% (95% CI, 39% to 67%) and 49% (CI, 35% to 63%), and IGF-1 levels were normal in 68% (CI, 54% to 82%) and 55% (CI, 40% to 70%) of patients receiving low- and high-dose octreotide, respectively. A substantial decrease in headache, amount of perspiration, joint pain, and finger circumference occurred in two thirds of the patients. The pituitary size was reduced in 19% (CI, 5% to 33%) and 37% (CI, 22% to 52%) of patients receiving 6 months of low- and high-dose octreotide, respectively. Ten percent and 13% of patients in each treatment group developed transient diarrhea; 10% and 14%, biliary sludge; and 6% and 18%, cholelithiasis, respectively. CONCLUSION: Octreotide effectively decreased GH and IGF-1 concentrations in 53% and 68% of patients, respectively. The higher dose resulted in increased frequency of tumor shrinkage but added no biochemical or clinical benefit.

Acromegaly↗
PubMed1992

The role of ion channels in insulin secretion.

Ion channels in beta cells regulate electrical and secretory activity in response to metabolic, pharmacologic, or neural signals by controlling the permeability to K+ and Ca2+. The ATP-sensitive K+ channels act as a switch that responds to fuel secretagogues or sulfonylureas to initiate depolarization. This depolarization opens voltage-dependent calcium channels (VDCC) to increase the amplitude of free cytosolic Ca2+ levels ([Ca2+]i), which triggers exocytosis. Acetyl choline and vasopressin (VP) both potentiate the acute effects of glucose on insulin secretion by generating inositol 1,4,5-trisphosphate to release intracellular Ca2+; VP also potentiates sustained insulin secretion by effects on depolarization. In contrast, inhibitors of insulin secretion decrease [Ca2+]i by either hyperpolarizing the beta cell or by receptor-mediated, G-protein-coupled effects to decrease VDCC activity. Repolarization is initiated by voltage- and Ca(2+)-activated K+ channels. A human insulinoma voltage-dependent K+ channel cDNA was recently cloned and two types of alpha 1 subunits of the VDCC have been identified in insulin-secreting cell lines. Determining how ion channels regulate insulin secretion in normal and diabetic beta cells should provide pathophysiologic insight into the beta cell signal transduction defect characteristic of non-insulin dependent diabetes (NIDDM).

Animals↗
PubMed1992

Cloning of a novel alpha 1-subunit of the voltage-dependent calcium channel from the beta-cell.

To study the molecular regulation of voltage-dependent Ca2+ channels (VDCCs) in the beta-cell, we have cloned a cDNA for the alpha 1-subunit from a hamster insulin-secreting cell line (HIT-T15). The cDNA (HCa3a) encodes a 1610-amino acid protein with four repeating membrane domains and an overall structure characteristic of other alpha 1-subunits. Although the cDNA shows a high degree of sequence homology (97%) with a rat brain alpha 1-subunit (RB alpha 1), the C-terminal 15 amino acids of HCa3a share no similarity with any cloned alpha 1 protein. High stringency Northern blot analysis revealed a single transcript of approximately 8.6 kilobases in HIT cells and hamster pancreas. A similarly sized species was detected in hamster brain, heart, and skeletal muscle. Using polymerase chain reaction and a primer set unique to HCa3a, this alpha 1 isoform was found to be expressed in islet cell lines derived from rat, mouse, and hamster. The HIT cell alpha 1-subunit is also expressed in discrete regions of the rat central nervous system, including the cortex, cerebellum, hypothalamus, and brain stem. The expression of two alpha 1 isoforms (HCa3a and cardiac) in the HIT cell underscores the possible complexity of VDCCs in the regulation of beta-cell signal transduction. With its widespread tissue distribution, HCa3a does not conform to the current classification system used for L-type VDCCs; this suggests that an alternative system of classification is required.

Amino Acid Sequence↗
PubMed1991

Somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry through voltage-dependent Ca2+ channels in the beta cell.

We tested the hypothesis that somatostatin (SRIF) inhibits insulin secretion from an SV40 transformed hamster beta cell line (HIT cells) by an effect on the voltage-dependent Ca2+ channels and examined whether G-proteins were involved in the process. Ca2+ currents were recorded by the whole cell patch-clamp method, the free cytosolic calcium, [Ca2+]i, was monitored in HIT cells by fura-2, and cAMP and insulin secretion were measured by radioimmunoassay. SRIF decreased Ca2+ currents, [Ca2+]i, and basal insulin secretion in a dose-dependent manner over the range of 10(-12)-10(-7)M. The increase in [Ca2+]i and insulin secretion induced by either depolarization with K+ (15 mM) or by the Ca2+ channel agonist, Bay K 8644 (1 microM) was attenuated by SRIF in a dose-dependent manner over the same range of 10(-12)-10(-7) M. the half-maximal inhibitory concentrations (IC50) for SRIF inhibition of insulin secretion were 8.6 X 10(-12) M and 8.3 X 10(-11) M for K+ and Bay K 8644-stimulated secretion and 1 X 10(-10) M and 2.9 X 10(-10) M for the SRIF inhibition of the K+ and Bay K 8644-induced rise in [Ca2+]i, respectively. SRIF also attenuated the rise in [Ca2+]i induced by the cAMP-elevating agent, isobutylmethylxanthine (1 mM) in the presence of glucose. Bay K 8644, K+ and SRIF had no significant effects on cAMP levels and SRIF had no effects on adenylyl cyclase activity at concentrations lower than 1 microM. SRIF (100 nM) did not change K+ efflux (measured by 86Rb+) through ATP-sensitive K+ channels in HIT cells. SRIF (up to 1 microM) had no significant effect on membrane potential measured by bisoxonol fluorescence. Pretreatment of the HIT cells with pertussis toxin (0.1 microgram/ml) overnight abolished the effects of SRIF on Ca2+ currents, [Ca2+]i and insulin secretion implying a G-protein dependence in SRIF's actions. Thus, one mechanism by which SRIF decreases insulin secretion is by inhibiting Ca2+ influx through voltage-dependent Ca2+ channels, an action mediated through a pertussis toxin-sensitive G-protein.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Sulfonylurea signal transduction.

In the pancreatic beta cells the proximal step in sulfonylurea signal transduction is the binding of these clinically important drugs to high-affinity receptors in the beta cell membrane. Using HIT cells as a model system, we have established an extremely close correlation between the affinity of binding of glyburide and its analog, iodoglyburide, and the activation of various steps in stimulus-secretion coupling--inhibition of 86Rb+ efflux, increase in [Ca2+]i resulting from gating of voltage-gated calcium channels by cell depolarization, and the exocytosis of insulin. Two different L-type channel cDNAs have been identified in an HIT cell library, one neuroendocrine in type and one more cardiac-like. A HIT cell membrane protein of Mr 140,000, which we believe to be the high-affinity sulfonylurea receptor, can be covalently linked to 5(125)-iodo-2-hydroxyglyburide by ultraviolet irradiation. The receptor has been solubilized and retains binding activity and the same rank order of displacement of the 5(125)-iodo-2-hydroxyglyburide as observed with the native receptor. The Mr 140,000 protein has been partially purified and the amino acid sequences of three proteolytic fragments have been used to design oligonucleotides to screen HIT cell cDNA libraries. Since the binding constant of glyburide or iodoglyburide is closely correlated with the ability of these compounds to inhibit the ATP-sensitive K+ channel, increase [Ca2+]i, and elicit insulin secretion, we have identified the Mr 140,000 protein as the sulfonylurea receptor. Expression of the cloned cDNA should allow us to test this hypothesis directly.

ATP-Binding Cassette Transporters↗
PubMed1991

Activation of alpha 2-adrenergic receptors decreases Ca2+ influx to inhibit insulin secretion in a hamster beta-cell line: an action mediated by a guanosine triphosphate-binding protein.

Activation of the sympathetic nervous system inhibits insulin secretion. We tested the hypothesis that activation of alpha 2-adrenergic receptors on the beta-cell by epinephrine or clonidine attenuates insulin release by an effect on the voltage-dependent Ca2+ channel (VDCC) and examined the role of G-proteins in this signal transduction pathway. Using a cultured SV40-transformed hamster beta-cell line (HIT cells) as a model system, we determined the effect of alpha 2-adrenergic agonists on insulin secretion, 86Rb+ efflux (a marker for K+ channel flux), and the free cytosolic Ca2+ level [( Ca2+]i) monitored in fura-2-loaded cells. In a dose-dependent manner, epinephrine and clonidine (10(-8)-10(-5)M) attenuated the increase in [Ca2+]i and insulin secretion induced by either K+ depolarization or stimulation of the VDCC with the agonist Bay K 8644. Epinephrine failed to affect the rise in [Ca2+]i induced by carbamylcholine, an agent that mobilizes intracellular Ca2+. Epinephrine also did not changes 86Rb+ efflux from HIT cells. The inhibitory effects of epinephrine were prevented by the alpha 2-adrenergic antagonist idazoxan, but were unaffected by the alpha 1-adrenergic antagonist phenoxybenzamine. Pretreatment of HIT cells with pertussis toxin (0.1 micrograms/ml) overnight abolished the inhibitory effects of epinephrine and clonidine on both [Ca2+]i and insulin secretion. These data suggest that one mechanism by which alpha 2-adrenergic agonists inhibit insulin secretion is by inhibiting Ca2+ influx through VDCC, an action that is mediated through a pertussis toxin-sensitive G-protein.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Molecular mechanisms of action of glyburide on the beta cell.

A high-affinity sulfonylurea receptor has been identified on the plasma membrane of the beta cell. The potent second-generation sulfonylureas, glyburide and glipizide, saturate the receptor in the low nM concentration range, whereas first-generation drugs bind to and saturate the receptor in the microM range. For each of the sulfonylureas, there is excellent quantitative agreement among the equilibrium binding constant (Kd), the half-maximal inhibition of potassium ion (K+) efflux (K0.5), and the half-maximal stimulation of insulin secretion (ED50), when these values are obtained from insulin-secreting cell lines or from isolated mouse pancreatic islets. The inhibition of K+ efflux by the sulfonylureas, coupled with the sulfonylurea inhibition of the activity of a specific adenosine triphosphate (ATP)-sensitive K+ channel embedded in the plasma membrane of whole cells or in excised membrane patches, suggests that the sulfonylurea receptor is this channel protein or a closely associated subunit. The activity of the ATP-sensitive K+ channel is also controlled by the insulin secretagogues, glucose and certain amino acids. These compounds must be metabolized to inhibit the channel activity and appear to do so by increasing the level of ATP or by increasing the ATP/adenosine diphosphate (ADP) ratio. ATP reduces channel activity by binding to a specific nucleotide-binding site on the cytoplasmic surface of the protein. There is a synergy between the action of glucose and that of the sulfonylureas. The sulfonylureas, for example, are better effectors of insulin secretion in the presence of glucose. Inhibition of the ATP-sensitive K+ channels results in depolarization of the plasma membrane and a subsequent influx of extracellular calcium ions through voltage-dependent calcium channels. An increase in the free intracellular calcium level is the signal, or "second messenger," that triggers exocytosis and the release of insulin. The sulfonylurea receptor has a molecular weight of 140,000 and can be solubilized by digitonin, retaining the same rank order of sulfonylurea binding affinities as the membrane-bound protein. Several laboratories are currently purifying the receptor and/or cloning the receptor gene.

Adenosine Triphosphate↗
PubMed1990

Molecular cloning of a novel splice variant of the alpha subunit of the mammalian Go protein.

We screened a HIT (hamster insulin-secreting tumor) cell cDNA library constructed in lambda gt11 with a Go-specific oligonucleotide probe and isolated six recombinant phages. The inserts of these phages encoded two forms of alpha o, called here alpha o1 and alpha o2. The deduced amino acid sequence of alpha o1 is identical in all of its 354 amino acids to that reported previously for rat and bovine alpha o; that of alpha o2, also of 354 amino acids, is identical to alpha o1 up to and including amino acid 248 and differs thereafter in 26 amino acids. At the nucleotide level, alpha o1 and alpha o2 are identical up to and including the second base of the codon that specifies amino acid 243 and differs thereafter in 88 nucleotides of the remaining open reading frame and has no similarity to alpha o1 in its 3'-untranslated region. We propose that alpha o1 and alpha o2 result as a consequence of alternative splicing of a single alpha o transcript. Northern analysis with specifically designed oligonucleotides indicates that both forms of alpha o are expressed in normal tissues, e.g. brain. After in vitro transcription and translation, the peptides encoded in the alpha o1 and alpha o2 cDNAs could be ADP-ribosylated by pertussis toxin in the presence of added beta gamma dimers. The count of distinct G proteins keeps increasing.

Adenosine Diphosphate Ribose↗
PubMed1990

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog.

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-"glyburide", has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values of 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).

ATP-Binding Cassette Transporters↗
PubMed1990

Ion channels and insulin secretion.

We review the role of ion channels in regulating insulin secretion from pancreatic beta-cells. By controlling ion permeability, ion channels at the membrane play a major role in regulating both electrical activity and signal transduction in the beta-cell. A proximal step in the cascade of events required for stimulus-secretion coupling is the closure of ATP-sensitive K+ channels, resulting in cell depolarization. Of particular relevance is the finding that this channel is directly regulated by a metabolite of glucose, which is the primary insulin secretagogue. In addition, this channel, or a closely associated protein, contains the sulfonylurea-binding site. Another K+ channel, the Ca2(+)-activated K+ channel, may be involved in cell repolarization to create homeostasis. Voltage-dependent Ca2+ channels are activated by cell depolarization and regulate Ca2+ influx into the cell. By controlling cytosolic free-Ca2+ levels ([Ca2+]i), these channels play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion. The link between a rise in [Ca2+]i and the terminal event of exocytosis is the least-understood aspect of stimulus-secretion coupling. However, phosphorylation studies have identified substrate proteins that may correspond to those involved in smooth muscle contraction, suggesting an analogy in the processes of stimulus secretion and excitation contraction. The advent of new methodology, particularly the patch-clamp technique, has fostered a more detailed characterization of the beta-cell ion channels. Furthermore, biochemical and molecular approaches developed for the structural analysis of ion channels in other tissues can now be applied to the isolation and characterization of the beta-cell ion channels. This is of particular significance because there appear to be tissue-specific variations in the different types of ion channels. Given the importance of ion channels in cell physiology, a knowledge of the structure and properties of these channels in the beta-cell is required for understanding the abnormalities of insulin secretion that occur in non-insulin-dependent diabetes mellitus. Ultimately, these studies should also provide new therapeutic approaches to the treatment of this disease.

Animals↗
PubMed1989

Catecholamine modulation of calcium currents in clonal pancreatic beta-cells.

The mechanisms by which norepinephrine and epinephrine activate alpha 2-adrenergic receptors and inhibit insulin release from the pancreatic beta-cell (19, 21, 23) are not yet clear but may involve modulation at several sites. Because intracellular calcium has been implicated in the secretory process, it has been suggested that catecholamines may inhibit secretion by blocking calcium influx, thus reducing the free cytosolic calcium concentration (23). The present study examines the effects of epinephrine, norepinephrine, and clonidine on calcium current in an SV40-transformed hamster beta-cell line (HIT cells). Under voltage-clamp conditions, calcium currents were reversibly inhibited by norepinephrine, epinephrine, and clonidine in the low nanomolar range. The effects were blocked by 1) the alpha 2-antagonist yohimbine, 2) preincubation of the cells with pertussis toxin (PTX), and 3) guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), the nonhydrolyzable GDP analogue that competitively inhibits the interaction of GTP with G proteins. In contrast, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused irreversible blockade by catecholamines. These effects could not be overcome by adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that the adenylate cyclase pathway is not involved in the G protein coupling with the channels. These studies show that catecholamines inhibit calcium currents in beta-cells through an alpha 2-adrenoreceptor PTX-sensitive G protein pathway and could inhibit insulin secretion by this mechanism.

Adenylate Cyclase Toxin↗
PubMed1989

Characterization of voltage-dependent Ca2+ channels in beta-cell line.

Although there is compelling pharmacological evidence based on Ca2+-channel antagonist studies suggesting that the voltage-dependent Ca2+ channels regulate insulin release, no direct comparison with Ca2+ currents exists. This is particularly important because of the recent demonstration in other cell types of one and possibly two Ca2+ channels that are insensitive to Ca2+-channel antagonists, the dihydropyridines and the phenylalkylamines. Using an SV40-transformed pancreatic beta-cell line (HIT cells), we determined how voltage-dependent Ca2+ channels are involved in stimulus-secretion coupling. Ca2+ currents were measured with the tight-seal technique for whole-cell recording. The cytosolic free-Ca2+ concentration ([Ca2+]i) was followed with the fluorescent probe Fura 2, and the measurements were compared with insulin secretion stimulated by depolarizing the cells with K+. The Ca2+ current contained two components: a rapidly decaying current activated at -50 to -40 mV that decayed with a time constant of 25 ms and a very slowly decaying component activated at -40 mV. Both components were sensitive to the Ca2+-channel antagonist nimodipine. There is excellent agreement in the concentration of nimodipine that inhibited Ca2+ and the increase in [Ca2+]i in response to K+ depolarization (IC50 of 15 and 6 nM, respectively). Nimodipine inhibited insulin release over a similar dose-response range with an IC50 of 1.5 x 10(-9) M. These studies indicate that the increase in [Ca2+]i in response to beta-cell depolarization can be accounted for by the influx of this ion through a single class of dihydropyridine-sensitive Ca2+ channels in the cell membrane.

Animals↗
PubMed1989

Effect of rise in cAMP levels on Ca2+ influx through voltage-dependent Ca2+ channels in HIT cells. Second-messenger synarchy in beta-cells.

With a glucose-responsive beta-cell line (HIT cells), we tested the hypothesis that the cytosolic free-Ca2+ level ([Ca2+]i) is an intracellular signal through which a rise in cyclic AMP (cAMP) levels is transmitted to potentiate glucose-stimulated insulin secretion. In these cells, glucose stimulates the acute release of insulin without increasing [Ca2+]i or altering cAMP content. Either forskolin or 3-isobutylmethylxanthine (IBMX) potentiated glucose-stimulated insulin secretion and increased cAMP levels. At either a submaximal glucose concentration or maximally stimulatory glucose concentration, both IBMX and forskolin triggered a rapid rise in [Ca2+]i (1.9- and 1.5-fold increase over basal levels, respectively). Similarly, glucagon stimulated a 1.3-fold increase in [Ca2+]i over basal levels. The effect on [Ca2+]i required glucose and was secondary to Ca2+ influx through voltage-dependent Ca2+ channels because it was blocked by either chelation of extracellular Ca2+ with EGTA or by the Ca2+-channel blockers verapamil and nimodipine. Verapamil also inhibited IBMX potentiation of glucose-stimulated insulin secretion and the IBMX-induced rise in [Ca2+]i in a dose-dependent manner with IC50s of 2 x 10(-5) and 4 x 10(-6) M, respectively. We conclude that in the beta-cell, a rise in cAMP levels increases Ca2+ influx through voltage-dependent Ca2+ channels and that this represents a mechanism by which cAMP potentiates glucose-stimulated insulin secretion in beta-cells.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Characterization of the sulfonylurea receptor on beta cell membranes.

Specific, high affinity sulfonylurea receptors were characterized on membranes of an insulin-secreting hamster beta cell line (HIT cells). Saturable binding of the sulfonylurea, [3H]glyburide, was linear up to 0.8 mg/ml membrane protein. Scatchard analysis of equilibrium binding data at room temperature indicated the presence of a single class of saturable, high affinity binding sites with a Kd of 0.76 +/- 0.04 nM and a Bmax of 1.09 +/- 0.13 pmol/mg protein, n = 9. The insulin secretory potency of glyburide, glipizide, tolbutamide, tolazamide, and carboxytolbutamide was compared to the ability of these ligands to displace [3H]glyburide from the sulfonylurea receptor. Tolbutamide, tolazamide, and glipizide demonstrated reasonable agreement with ED50 values of 15 microM, 3 microM, and 30 nM and Ki values of 25.3 microM, 7.2 microM, and 45 nM, respectively. The inactive tolbutamide metabolite, carboxytolbutamide, at the highest concentration tested, only partially displaced [3H]glyburide from the receptor and was a very poor secretagogue. At 37 degrees C the affinity of [3H]glyburide binding, Kd = 2.0 nM, was similar to the ED50 of 5.5 nM when the free glyburide concentrations were corrected for binding of the drug to albumin. These studies suggest that sulfonylureas initiate their biologic effect through a high affinity, specific interaction with sulfonylurea receptors on the beta cell membrane.

ATP-Binding Cassette Transporters↗
PubMed1988

Sulfonylurea receptors, ion channels, and fruit flies.

Recent studies have identified a high-affinity receptor on the plasma membrane of the beta-cell that is specific for all of the sulfonylureas. The most potent second-generation drugs, glyburide and glipizide, bind to the receptor and trigger insulin release at nanomolar concentrations. The affinity to the receptor-ligand interaction of all sulfonylureas correlates with their potency as insulin secretagogues, further implicating receptor occupancy with signal transduction. These drugs also inhibit the electrical activity of ATP-sensitive K+ channels and K+ efflux through these channels. The channels are also closed by the metabolism of the major insulin secretagogues, glucose and the amino acids, which signal insulin release by increasing the ATP level or the [ATP]-to-[ADP] ratio on the cytoplasmic side of the channel. Based on the channel number and the amount of K+ current they pass, it is possible to calculate that these channels control the resting membrane potential of the beta-cell. Inactivation of the ATP-inhibitable K+ channel results in a fall in the resting membrane potential, cell depolarization, and influx of extracellular Ca2+ through the voltage-dependent Ca2+ channel. The rise in intracellular free Ca2+ level triggers exocytosis. Thus, it is now possible to link either a stimulus from the metabolism of insulin secretagogues or the sulfonylureas to ionic and electrical events that elicit insulin release. These data also suggest that the sulfonylurea receptor or a closely associated protein is an ATP-sensitive K+ channel.

ATP-Binding Cassette Transporters↗
PubMed1987

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells.

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.

Animals↗
PubMed1987

Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes.

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca2+ are mediated by the Ca2+-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca2+-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca2+-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an 125I-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Ca2+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100 degrees C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased 125I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52,000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membrane. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggest a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.

Animals↗