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Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin for insulin secretion from the pancreatic beta-cell, but its mechanism of action has not been established. It interacts with specific cell-surface receptors, generates cAMP, and thereby activates protein kinase A (PKA). Many changes in pancreatic beta-cell function have been attributed to PKA activation, but the contribution of each one to the secretory response is unknown. We show here for the first time that GLP-1 rapidly released free fatty acids (FFAs) from cellular stores, thereby lowering intracellular pH (pHi) and stimulating FFA oxidation in clonal beta-cells (HIT). Similar changes were observed with forskolin, suggesting that stimulation of lipolysis was a function of PKA activation in beta-cells. Triacsin C, which inhibits the conversion of FFAs to long-chain acyl CoA (LC-CoA), enhanced basal FFA efflux as well as GLP-1-induced acidification and efflux of FFAs from the cell. Increasing the concentration of the lipase inhibitor orlistat progressively and largely diminished the increment in secretion caused by forskolin. However, glucose-stimulated secretion was less inhibited by orlistat and only at the highest concentration tested. Because the acute addition of FFAs also increases glucose-stimulated insulin secretion, these data suggest that the incretin function of GLP-1 may involve a major role for lipolysis in cAMP-mediated potentiation of secretion.

The initiation of insulin release from rat islet beta cells relies, in large part, on calcium influx through dihydropyridine-sensitive (alpha1D) voltage-gated calcium channels. Components of calcium-dependent insulin secretion and whole cell calcium current, however, are resistant to L-type channel blockade, as well as to omega-conotoxin GVIA, a potent inhibitor of alpha1B channels, suggesting the expression of additional exocytotic calcium channels in the islet. We used a reverse transcription-polymerase chain reaction-based strategy to ascertain at the molecular level whether the alpha1A calcium channel isoform was also present. Results revealed two new variants of the rat brain alpha1A channel in the islet with divergence in a putative extracellular domain and in the carboxyl terminus. Using antibodies and cRNA probes specific for alpha1A channels, we found that the majority of cells in rat pancreatic islets were labeled, indicating expression of the alpha1A channels in beta cells, the predominant islet cell type. Electrophysiologic recording from isolated islet cells demonstrated that the dihydropyridine-resistant current was sensitive to the alpha1A channel blocker, omega-agatoxin IVA. This toxin also inhibited the dihydropyridine-resistant component of glucose-stimulated insulin secretion, suggesting functional overlap among calcium channel classes. These findings confirm the presence of multiple high voltage-activated calcium channels in the rat islet and implicate a physiologic role for alpha1A channels in excitation-secretion coupling in beta cells.

One hundred and ninety-seven members of 28 kindreds with multiple endocrine neoplasia type 2A (MEN 2A) were screened for RET proto-oncogene exon 10 and 11 mutations. Seventy-one known affected individuals had mutations of codons 609, 618, 620 or 634, whereas 53 unaffected individuals had no abnormalities. Nineteen out of 54 individuals of unknown status, mostly children, had RET mutations. Four of these children had thyroidectomy based on this analysis and were found to have C-cell abnormalities. We identified one false negative mutation analysis because of a codon 691 polymorphism. We conclude that RET mutational analysis is a cost-effective and accurate method for determination of gene carrier status in MEN 2A.

Sulfonylureas are a class of drugs widely used to promote insulin secretion in the treatment of non-insulin-dependent diabetes mellitus. These drugs interact with the sulfonylurea receptor of pancreatic beta cells and inhibit the conductance of adenosine triphosphate (ATP)-dependent potassium (KATP) channels. Cloning of complementary DNAs for the high-affinity sulfonylurea receptor indicates that it is a member of the ATP-binding cassette or traffic ATPase superfamily with multiple membrane-spanning domains and two nucleotide binding folds. The results suggest that the sulfonylurea receptor may sense changes in ATP and ADP concentration, affect KATP channel activity, and thereby modulate insulin release.

Glucagon-like peptide-I (GLP-I) is a potent incretin hormone and is considered as a new therapeutic tool in the treatment of diabetes mellitus. This study was designed to precisely characterize the binding behavior and activation of the recombinant GLP-I receptor against naturally occurring ligands of the glucagon/VIP/secretin peptide hormone family. CHO-cells were stably transfected with a plasmid containing a cDNA encoding for the rat GLP-I receptor. Northern blot analysis with this cDNA showed a single band of 2.7 kb in CHO cells, while in RINm5F cells, three bands of 2.7, 3.4, and 3.6 kb were specifically labelled. In receptor-binding studies 125I-GLP-I was displaced by GLP-I and weakly by PHI and oxyntomodulin but not by helodermin, helospectin I, helospectin II, secretin, VIP, and PACAP-38. Intracellular cAMP generation was stimulated by GLP-I, PHI, and oxyntomodulin. Helodermin, helospectin I, helospectin II, secretin, VIP, and PACAP-38 were not able to displace 125I-GLP-I from its receptor or to stimulate intracellular cAMP production. This data shows that the GLP-I receptor is characterized by a high ligand specificity.

Significant advances have been made in the understanding of the pathophysiology and the ability to effectively screen for and treat medullary thyroid carcinoma. The parafollicular cells, or C-cells, are the cell of origin for medullary thyroid carcinoma. C-cell hyperplasia is a premalignant disease that progresses rapidly to medullary thyroid carcinoma. C-cells produce calcitonin, which serves as a marker to prospectively screen patients for C-cell disease. One major concern in screening for this disease has been the incidence of false positive results. This problem is addressed in light of new, more stringent criteria for the diagnosis of C-cell hyperplasia. The association of hyperparathyroidism with MEN 2 is discussed with evidence that thyroidectomy of C-cell disease may affect the incidence of parathyroid disease.

Sulfonylureas interact with specific, high affinity receptors on the pancreatic beta-cell to close ATP-sensitive K+ channels, depolarize the cell, activate Ca2+ influx through voltage-dependent Ca2+ channels, and trigger insulin secretion. We tested the hypothesis that sulfonylureas promote glucose uptake into 3T3-L1 cells or isolated rat adipocytes by similar mechanisms. Using 125I-labeled 5-iodo-2-hydroxyglyburide and either equilibrium binding or photoaffinity labeling, a high affinity sulfonylurea receptor was not found on plasma membranes of either the 3T3-L1 cells or rat adipocytes. Furthermore, glyburide did not inhibit 86Rb+ efflux (a marker for ATP-sensitive K+ channel conductance), increase free cytosolic calcium in adipocytes or 3T3-L1 cells, or increase basal or insulin-stimulated glucose uptake into 3T3-L1 cells or rat adipocytes. Parallel studies using a hamster insulin-secreting tumor cell line (HIT cells) easily demonstrated both the receptor and biological effects of glyburide on free cytosolic calcium and insulin secretion. Thus, rat adipocytes and 3T3-L1 cells do not possess the high affinity sulfonylurea receptor or respond to glyburide alone. We conclude that the antidiabetogenic effects of sulfonylureas are not mediated by a direct action of sulfonylureas to increase glucose uptake into adipose tissue and suggest that the major locus for sulfonylurea action is the beta-cell.

Glucagon-like peptide-I (GLP-I) is a potent insulinotropic peptide that mediates its actions at pancreatic B-cells via specific receptors. In the present study we stably expressed the rat B-cell GLP-I receptor in CHO cells and studied binding characteristics and receptor activation utilizing the naturally occurring receptor agonist GLP-I(7-36)-amide (GLP-I), the proglucagon-derived GLP-I-related peptide oxyntomodulin, the GLP-I receptor agonist exendin-4, and the specific antagonist exendin(9-39). The potencies to displace [125I]GLP-I from the receptor were GLP-I > exendin-4 > exendin(9-39) > oxyntomodulin, and to displace [125I]exendin-4 GLP-I = exendin-4 > exendin(9-39) > oxyntomodulin. cAMP production was stimulated equally by GLP-I and exendin-4. Oxyntomodulin was less potent to stimulate cAMP generation. Exendin(9-39) blocked the stimulatory action of GLP-I and exendin-4 on cAMP production, but not that of oxyntomodulin. This study shows that GLP-I and exendin-4 are potent agonists at the transfected rat B-cell GLP-I receptor whereas oxyntomodulin is only a weak GLP-I receptor agonist. Furthermore, exendin(9-39) is a potent GLP-I receptor antagonist. This peptide is a valuable tool to further study the physiological actions of GLP-I.

Truncated forms of glucagon-like peptide-1 are the most potent endogenous stimuli of insulin secretion and have powerful antidiabetogenic effects. To determine the structure and coupling mechanisms of the human GLP-1 receptor we have isolated two pancreatic islet cDNAs, encoding the 463 amino acid receptor and differing mainly in their 3' untranslated regions. The deduced amino acid sequence is 90% homologous with the rat GLP-1 receptor. Northern blot analysis shows expression of a single 2.7 kb transcript in pancreatic tissue. When expressed in COS-7 cells the recombinant receptor conferred specific, high affinity GLP-1(7-37) binding. GLP-1(7-37) increased intracellular cAMP in a concentration dependent manner and caused an increase in the free cytosolic calcium ([Ca2+]i) from an intracellular pool, characteristic of phospholipase C (PLC) activation. Thus, like the structurally related glucagon and parathyroid hormone receptors, the human GLP-1 receptor can activate multiple intracellular signaling pathways including adenylyl cyclase and PLC. Knowledge of the GLP-1 receptor structure will facilitate the development of receptor agonists and elucidation of the important role of GLP-1 in normal physiology and disease states.

In man, glucagon-like peptide-I-(7-37) [GLP-I-(7-37)] is the most potent endogenous insulin-stimulating hormone. Although GLP-I-(7-37)-stimulated insulin secretion from the beta-cell is associated with an increase in cAMP accumulation, little is known about the signal transduction pathways used by this peptide. Using a cDNA encoding a high affinity rat GLP-I-(7-37) receptor [Kd = 4.1 nM for GLP-I-(7-37); Kd = 1 microM for GLP-I-(1-36) amide] expressed in a monkey kidney cell line (COS-7), we have demonstrated that the receptor is not only coupled to adenylyl cyclase, but is associated with an increase in the free cytosolic calcium level ([Ca2+]i). GLP-I-(7-37) increased both cAMP and [Ca2+]i in a dose-dependent manner and with equal potency (ED50 = 2.0 nM). The major source of the increased [Ca2+]i was found to be through the release of intracellular pools of Ca2+ associated with an increase in phosphoinositol turnover. Northern blot hybridization studies demonstrated that the GLP-I-(7-37) receptor gene was expressed in relatively high abundance in pancreatic islets and lung, but was also expressed at lower levels in the brain, liver, kidney, and skeletal muscle. This study establishes that a single GLP-I receptor species can mediate the effects of GLP-I-(7-37) through multiple G-protein-coupled signaling pathways, including the adenylyl cyclase system, phospholipase-C, and changes in [Ca2+]i.

The mechanisms underlying the glucose dependence of arginine vasopressin (AVP)-stimulated insulin secretion were examined in a hamster insulin-secreting cell line (HIT cells). At 1.67 mM glucose, 100 nM AVP stimulated biphasic changes in free cytosolic Ca2+ ([Ca2+]i) and insulin secretion. The initial spike of [Ca2+]i came from an intracellular pool and was accompanied by parallel changes in the levels of inositol 1,4,5-trisphosphate. The following sustained increase in [Ca2+]i was associated with membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels. The rapid phase of insulin secretion and the [Ca2+]i spike were resistant to the Ca2+ channel blocker nimodipine, whereas the sustained insulin secretion and the protracted increase in [Ca2+]i were inhibited by nimodipine. Thus, biphasic increases in [Ca2+]i mediated the biphasic insulin secretory pattern. In the absence of glucose, 100 nM AVP triggered a transient smaller spike in [Ca2+]i, but did not stimulate membrane depolarization, Ca2+ influx, or insulin secretion. However, the increase in inositol 1,4,5-trisphosphate was similar to that seen at 1.67 mM glucose. Both the AVP-induced [Ca2+]i spike and sustained [Ca2+]i increase were augmented by glucose. We concluded that the initial AVP receptor-mediated activation of phospholipase-C is not altered by glucose, but both intracellular Ca2+ release and extracellular Ca2+ influx through voltage-dependent Ca2+ channels triggered by AVP are glucose dependent and explain the sensitivity of AVP-stimulated insulin release to this metabolite.

Using the glucose-responsive hamster beta-cell line (hamster insulin tumor cells), we examined the cellular mechanisms by which gastric inhibitory polypeptide (GIP) and glucagon-like peptide I(7-37) (GLP-I) potentiate glucose-stimulated insulin secretion. Glucose alone increased insulin secretion and increased the free cytosolic calcium levels ([Ca2+]i) without altering cAMP content. When added to glucose-stimulated cells, GIP and GLP-I increased cAMP levels and further increased insulin secretion. At 4 mM but not 0.4 mM glucose, both peptides triggered a dose-dependent rise in [Ca2+]i with ED50s of 0.4 and 0.2 nM for GIP and GLP-I, respectively. The increase in [Ca2+]i was blocked by either chelation of extracellular Ca2+ with EGTA or nimodipine, the voltage-dependent Ca2+ channel blocker. Nimodipine also inhibited the potentiation of glucose-stimulated insulin secretion by GIP and GLP-I without inhibition of the stimulatory effect of these two peptides on cAMP accumulation. Neither peptide altered phosphoinositide metabolism, further underlining that the mobilization of intracellular Ca2+ from endoplasmic reticulum is not involved in the GIP and GLP-I signal transduction pathways. This study establishes that GIP and GLP-I potentiate glucose-stimulated insulin secretion by increasing extracellular Ca2+ influx through voltage-dependent Ca2+ channels.

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.

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).

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.

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.

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.

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.

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).

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.

To determine if serum prolactin levels were correlated with the level of depression in an ambulatory medical clinic population, prolactin was measured by immunoassay in all new medical patients completing the Langer Scale and the Popoff Index of Depression. Thirty-four of 71 patients (48%) were found to be depressed with a positive Popoff Scale. There was no difference in the prolactin levels in the depressed and nondepressed patients, who were matched for age and sex. Thus, depression, as seen in an ambulatory medical population, is not associated with increased prolactin release.

We have previously shown in a glucose-responsive insulin-secreting beta-cell line (HIT cells) that a cell membrane-depolarizing concentration of K+ causes an influx of extracellular Ca2+ that elevates the free cytosolic Ca2+ concentration ([Ca2+]i) and activates insulin secretion. To identify the cellular mechanisms that couple Ca2+ influx to exocytosis, studies of protein phosphorylation were conducted. HIT cells were loaded with 32P and stimulated with either 40 mM K+ or 19.4 mM glucose, both of which trigger the immediate release of insulin. Phosphoproteins were examined with two-dimensional polyacrylamide gel electrophoresis and autoradiography. With addition of 40 mM K+, after the rise in [Ca2+]i, proteins of Mr 17,500, 20,000, and 24,000 were rapidly and specifically phosphorylated. The 17,000-Mr protein showed maximum phosphorylation by 1 min, whereas phosphorylation of the 24,000- and 20,000-Mr proteins continued to increase at 5 min. Despite stimulating the immediate release of insulin, high glucose did not result in a change in [Ca2+]i or the phosphorylation of the three Ca2+-dependent phosphoproteins. Insulin secretion, the increase in [Ca2+]i, and phosphorylation of the proteins by 40 mM K+ was inhibited by 100 microM verapamil, an organic Ca2+-channel-blocking drug. The rapid phosphorylation of three proteins, after a rise in [Ca2+]i and the coordinate inhibition of insulin secretion and phosphorylation, are consistent with the hypothesis that Ca2+ enters the beta-cell through voltage-dependent Ca2+ channels, regulating insulin release by effects on protein phosphorylation. However, glucose activates the immediate release of insulin by another, as yet undefined, mechanism.

Neuroendocrine function in two women with galactorrhea-amenorrhea arising from abnormalities in the PRL reflex arc was compared to that of normal women. Basal gonadotropins were lower than normal, and one patient lacked episodic secretion of LH; however, the serum gonadotropin rise after iv LRH was in the normal range in both patients. Mean basal PRL levels were slightly elevated in one patient and were normal in the other, and the PRL levels after TRH, chlorpromazine, and levodopa testing were similar to those seen in normal women. Breast stimulation did not increase PRL levels in either patient. PRL levels fell with bromergocryptine therapy, galactorrhea ceased, and normal menses resumed. These studies indicate that chronic afferent impulses originating in the PRL reflex arc can result in galactorrhea and amenorrhea and that bromergocryptine therapy in such patients can restore normal menses.

Tests of prolactin regulation in the galactorrhea-amenorrhea syndrome were compared in 18 patients with normal pituitary fossae, seven patients with prolactin-secreting adenomas, and eight normal women. Mean basal prolactin was highest in patients with adenomas and was elevated in those with normal fossae when compared with normal subjects (278 versus 73 versus 10.2 ng/ml). Levodopa, water loading, or luteinizing hormone-releasing hormone testing were of no predictive value in the diagnosis of adenoma. Some patients with adenomas show a greater prolactin response after administration of thyrotrophin hormone-releasing hormone (TRH) than of chlorpromazine, whereas these responses are usually similar in patients with normal fossae. A mean basal prolactin level above 150 ng/ml or an increase of more than 100 ng/ml after TRH administration in a patient with hyperprolactinemia unresponsive to chlorpromazine stimulation strongly suggests a prolactin-secreting tumor. However, because some patients with tumor have prolactin levels below 150 ng/ml, or do not respond to TRH stimulation, or both, functional studies alone cannot permit the diagnosis of all adenomas before the appearance of radiographic changes.

Growth hormone production by a rat pituitary tumor cell line (GH1) was measured during lag, exponential, and plateau phases of growth in different culture media. Growth hormone secretion was low during lag and early exponential phase; it increased late in the exponential phase and continued to increase during the plateau phase. This biphasic pattern of growth hormone production was observed in all media and sera utilized. Both the doubling time and growth hormone production were influenced by the choice of media and sera. In addition, the length of time in culture affected the growth fraction with passage level 40 GH1 cells having a 79% growth fraction, whereas the growth fraction of passage level 100 cells was 95%. Using the population doubling time as a criterion for a choice of medium, F-10 medium supplemented with 20% fetal bovine serum consistently yielded the most rapid doubling time (32 hr), whereas Dulbecco's MEM supplemented with 15% horse serum and 2.5% fetal bovine serum yielded the greatest plateau cell density. Growth hormone secretion and the population doubling times were directly related to culture conditions including length of time in culture, choice of tissue culture media, choice of sera, and the phase of cell growth (lag, exponential or plateau).

We gave pergolide mesylate, a new long-acting ergot derivative with dopaminergic properties, to 47 patients with hypersecretion of prolactin or growth hormone. Single doses produced long-lasting reductions of serum prolactin levels; after 24 hours, the values remained depressed at a mean of 28.8 per cent of the base-line value. Among 41 patients (22 women and 19 men) with hyperprolactinemia who took pergolide for three months or more, prolactin levels fell to normal in 37 and remained slightly elevated in 2. In the two patients in whom the levels fell to only 38 to 52 per cent of base line, treatment was regarded as a failure. The level of growth hormone fell to a mean of 52.8 per cent of base line in patients with acromegaly who were taking 100 micrograms of pergolide per day. Among patients for whom adequate CT scans were available, definite tumor shrinkage occurred in 10 of 13 with macroadenomas and definite or probable shrinkage in 5 of 9 with microadenomas. Menses returned in 76 per cent of treated women and testosterone levels rose in 10 of 14 men. We conclude that pergolide reduces hypersecretion and shrinks most prolactin-secreting macroadenomas. In some patients long-term pergolide therapy may be superior to surgery and x-ray treatment.

The primary care physician should educate all diabetics regarding the roles of diet, exercise, and insulin therapy in controlling the course of their disease, as well as the potential complications, including retinopathy. In type I diabetes, blood glucose levels should be controlled as rigidly as possible with multiple insulin injections and self-monitoring of glucose level. Hypertension should be aggressively treated in all diabetics. Yearly eye examination in patients with type I diabetes is important, and ophthalmologic referral should be considered after the disease has been present for five to ten years, with the first evidence of retinopathy, at puberty, during pregnancy, or if ocular symptoms occur. In patients with type II diabetes an ophthalmologist should make an evaluation as soon as the blood glucose level is controlled. Through use of these steps, the risk of blindness in diabetics will be minimized.

Forty-two patients with elevated serum prolactin were treated in a randomized, open-label trial with the conventional ergot bromocriptine, or a new ergot pergolide. Before treatment, the patients underwent thorough endocrine evaluation and computed tomographic scan. All patients had prolactin levels greater than 25 ng/ml, and 27 patients had a pituitary mass. Follow-up studies performed after 6 months of treatment showed both drugs effectively reduced prolactin levels to normal, though pergolide effects were more rapid. There was no change in the contents of the pituitary fossa in the 10 patients with hyperprolactinemia but without pituitary mass. Sixty percent of patients with pituitary mass had diminution of tumor size. Pergolide appears to be an effective medical treatment for hyperprolactinemia and pituitary tumor and offers a possible alternative to bromocriptine and surgical treatment.

Actin-binding proteins were assayed in various tissues using an 125I-actin overlay procedure. Four major G actin-binding proteins of 90000, 65000, 58000 and 40000 Mr have been identified. The 90K protein is present in all tissues and binds labelled actin in a calcium-sensitive manner with binding increasing 3-4-fold in the presence of Ca2+. The distribution of the 58K and 65K protein which are not Ca2+-sensitive was more variable. These proteins were present in different ratios in different tissues. 125I-actin binding to all four actin-binding proteins is specific and can be displaced by preincubation of the gels with unlabelled actin. The interaction of actin with these proteins does not appear to involve ionic forces, since binding is not diminished by varying the salt concentration. Skeletal muscle glycolytic enzymes, the lens crystallins and the histones also bind 125I-actin. This binding cannot be displaced by preincubation with unlabelled actin and is presumably non-specific. The calcium sensitivity of two highly purified actin-binding proteins, the 90K human platelet protein and villin was compared using 125I-actin. The platelet 90K protein binds actin at less than 10(-7) M free calcium, but detectable binding to villin does not occur below 10(-6) M free calcium. The ubiquity of these actin-binding proteins is clear and we conclude that the calcium-sensitive 90K actin-binding protein in all of these tissues is the same as the platelet protein.

Release of CHO-K1 cells from plateau or stationary phase and reentry into the cell cycle is specifically and reversibly blocked at two distinct sites by the anticalmodulin drug W13. The first block occurs early during release while the cells are still at G0/G1, whereas the second occurs later in reentry during early S phase. As determined by radioimmunoassay, calmodulin levels undergo changes at three distinct steps in plateau-phase entry and release. First, the entry of exponentially growing cells into plateau phase is accompanied by an increase in the calmodulin level. The second change is a reduction in the calmodulin content of cells within the first hour following release from plateau phase. The third change is the subsequent increase in calmodulin levels, which precedes entry of the cells into S phase. Analysis of calmodulin mRNA levels by dot-blot hybridization demonstrates that the changes in calmodulin protein are preceded by changes in calmodulin mRNA. Furthermore, whereas a decrease in CaM mRNA is observed within the first hour following plateau release, no such decrease is observed for beta-actin mRNA, suggesting that this decrease may be selective for calmodulin. This selectivity is further substantiated by the fact that identical changes in calmodulin and calmodulin mRNA are observed in cells released from plateau by two different techniques. Taken together, these data suggest that calmodulin may play an important role in the reentry of cells into the cell cycle.

Chlorpromazine is frequently administered to patients with hyperprolactinemia to stimulate an increase in the serum levels of prolactin. A patient with a prolactin secreting adenoma is described in whom pituitary apoplexy developed in association with a hypotensive episode following the administration of 25 mg of chlorpromazine. Prolactin levels fell from more than 2,000 ng/ml to 340 ng/ml following infarction of the pituitary tumor. Pituitary apoplexy should be considered as a rare complication of chlorpromazine stimulation in a patient with a pituitary tumor.

Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.

We have established somatic cell hybrids by fusing cells from two human insulinomas with an established murine cell line LMTK- Cl1D. After selection of the hybrids, the media were analyzed and found to contain insulin and human C-peptide immunoreactive material. The newly synthesized material was further characterized by pulse labeling and immunoprecipitation, and shown in five hybrid lines on Sephadex G-50 chromatography to have a size similar to proinsulin. The hybrids produced the apparent proinsulin-like material for up to 7 mo. Chromosome composition of the hybrids was determined by isozyme analysis and banding techniques. Chromosome 11, which previously has been assigned the insulin gene using cDNA probes, was identified in the hybrids producing proinsulin-like material. However, the retention of this chromosome did not always assure the production of hormone. This independent technique has confirmed the localization of the insulin gene to chromosome 11 and offers the opportunity of studying insulin processing and developing continuous insulin-producing cell lines.

Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.

An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.

Serum prolactin (PRL) and thyrotropin (TSH) levels were measured after iv administration of 200 microng of synthetic thyrotropin-releasing hormone (TRH) in 20 normal women ages 18 to 34. Ten women received TRH on days 7 to 8 of the menstrual cycle and 10 women received TRH on days 21-22. Although there was no difference in the dose of TRH relative to body weight in the two groups of women, the peak PRL level after TRH stimulation was greater in the women studied on day 21-22 (48.5+/-5.7 ng/ml, mean+/-SE) than on day 7-8 (35.2+/-4.2 ng/ml) of the cycle (P less than 0.05). In contrast, TSH rose to a greater degree in the preovulatory phase (13.8+/-1.8 micronU/ml) than the luteal phase (7.7+/-0.7 micronU/ml of the cycle (P less than .01). Studies of the PRL and TSH response after TRH administration should take the phase of the menstrual cycle into account.

Calmodulin concentrations were measured in isolated hamster islets or in a cloned rat insulin-secreting cell line RIN-m-5F treated with high glucose. There was no change in the cellular calmodulin content of either islets or RIN-m-5F cells despite increases of insulin concentrations in the media. Treatment of cells with the anti-calmodulin drug W13 inhibited insulin-stimulated glucose release, whereas a small effect on insulin accumulation in the media was observed with W12, the dechlorinated, less active analogue of W13. At the lowest dose tested (30 microM) the effect of W13 on insulin accumulation in the media was completely reversible. To further investigate the possible role of calmodulin in insulin secretion, calmodulin-binding proteins in subcellular fractions of the RIN-m-5F cells were identified using a gel overlay technique. Ca2+-dependent binding of 125I-calmodulin was observed to cytosolic proteins with apparent Mr = 125, 110, 56, 52, and 34 k (kilodalton). This binding was completely displaceable with unlabeled calmodulin, whereas only partial displacement was observed when the homologous Ca2+ binding protein of skeletal muscle, troponin C, was used. Four proteins with Mr similar to the histones bind calmodulin in a Ca2+-independent manner. 125I-calmodulin:calmodulin binding protein interactions were inhibited in a dose-dependent manner by the anti-calmodulin drug, W13. Little effect was observed with the analogue W12.(ABSTRACT TRUNCATED AT 250 WORDS)