Stimulus-secretion coupling of parathyroid hormone release: studies of 45Ca and 86Rb fluxes

Pieces of rat parathyroid glands were used to study fluxes of 45Ca and 86Rb. The uptake of 45Ca increased with the extracellular Ca2+ concentration up to at least 5 mM. A rise of extracellular Ca2+ had dual effects on 45Ca efflux in terms of an initial stimulation and a subsequent inhibition. However, K+ depolarization neither affected the uptake nor the efflux of 45Ca indicating a lack of voltage-dependent Ca2+ channels. The depolarization obtained with exposure to Ca2+ cannot be attributed to a decreased K+ permeability, since the 86Rb concentrating ability diminished and the efflux of the isotope increased when parathyroid pieces were exposed to a raised Ca2+ concentration. A stimulation of 86Rb efflux by the Ca2+ ionophore A-23187 indicated that the parathyroid cells possess a K+ permeability activated by cytoplasmic Ca2+. It is suggested that Ca2+ fluxes through channels sensitive to activation by Ca2+ are important both for the membrane potential and the cytoplasmic Ca2+ activity.     

Amounts and distribution of intracellular magnesium and calcium in pancreatic beta-cells

beta-Cell-rich pancreatic islets were incubated for 60-120 min in the presence of 1 mM or 20 mM glucose and analysed with regard to their contents of magnesium and calcium and how these elements were distributed among subcellular fractions. The islets contained 42 mmol magnesium per kg protein with as much as 70 mmol per kg protein in the microsomal fraction. Both the total amount and intracellular distribution of magnesium remained unaffected after raising the glucose concentration of the incubation medium. The islet content of calcium was twice as high as that of magnesium, the mitochondria and secretory granules accounting for most of the calcium in the sedimentable fractions. In both organelles a substantial fraction of calcium was exchangeable as indicated from the incorporation of 45Ca during 90 min of incubation of the islets. When raising the glucose concentration to 20 mM the percentage exchange of calcium increased from 10 to 27 in the mitochondria and from 13 to 28 in the secretory granules. The glucose stimulation of 45Ca uptake was not associated with a statistically significant increase in the total amounts of calcium. However, in addition to stimulating calcium/calcium exchange, it cannot be excluded that glucose also induces a net accumulation of intracellular calcium in the beta-cells.     

Caffeine inhibits cytoplasmic Ca2+ oscillations induced by carbachol and guanosine 5′-O-(3-thiotriphosphate) in hyperpolarized pancreatic ß-cells

The effects of caffeine on cytoplasmic Ca²⁺ oscillations induced by carbachol and guanosine 5-O-(3-thiotriphosphate) (GTP--S) were studied in individual mouse pancreatic ß-cells clamped at a hyperpolarized potential. Addition of 10 mM caffeine did not affect the cytoplasmic Ca²⁺ concentration ([Ca²⁺]₁) in ß-cells exposed to 20 mM glucose and hyperpolarized with diazoxide. Under similar conditions 100 M carbachol induced a typical response with a marked [Ca²⁺]_i peak followed by a lower sustained elevation. Irrespective of whether 10 mM caffeine was present, there were [Ca²⁺]_i transients with frequencies of 1–5/min superimposed on the sustained phase in 50–60% of the cells. In previously non-exposed cells the introduction of 10 mM caffeine caused temporary lowering of the sustained phase with disappearance of the transients. Subsequent omission of caffeine in the continued presence of carbachol caused a marked [Ca²⁺]_i peak followed by reappearance of the [Ca²⁺]_i, transients. However, in cells oscillating in the presence of caffeine its omission caused disappearance of the transients. In this case reintroduction of caffeine restored the transients.In cells kept at –70 mV by a patch pipette containing 100 M GTP--S and 3 mM Mg-ATP there were [Ca²⁺]_i transients with frequencies of 0.5–2.5/min. These transients were sufficiently pronounced to activate repetitively a K⁺ current. Addition of 10 mM caffeine caused disappearance of the [Ca²⁺]_i transients or reduction of their amplitudes and frequencies.The results indicate that caffeine does not activate Ca²⁺-induced Ca²⁺ release in hyperpolarized ß-cells but inhibits the Ca²⁺-mobilizing effect of inositol 1,4,5-trisphosphate. Correspondence to: E. Gylfe at the above address     

Glucose inhibits glucagon secretion by a direct effect on mouse pancreatic alpha cells

Aims/hypothesis The mechanisms by which glucose regulates glucagon release are poorly understood. The present study aimed to clarify the direct effects of glucose on the glucagon-releasing alpha cells and those effects mediated by paracrine islet factors. Materials and methods Glucagon, insulin and somatostatin release were measured from incubated mouse pancreatic islets and the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) recorded in isolated mouse alpha cells. Results Glucose inhibited glucagon release with maximal effect at 7 mmol/l. Since this concentration corresponded to threshold stimulation of insulin secretion, it is unlikely that inhibition of glucagon secretion is mediated by beta cell factors. Although somatostatin secretion data seemed consistent with a role of this hormone in glucose-inhibited glucagon release, a somatostatin receptor type 2 antagonist stimulated glucagon release without diminishing the inhibitory effect of glucose. In islets exposed to tolbutamide plus 8 mmol/l K⁺, glucose inhibited glucagon secretion without stimulating the release of insulin and somatostatin, indicating a direct inhibitory effect on the alpha cells that was independent of ATP-sensitive K⁺ channels. Glucose lowered [Ca²⁺]_i of individual alpha cells independently of somatostatin and beta cell factors (insulin, Zn²⁺ and γ-aminobutyric acid). Glucose suppression of glucagon release was prevented by inhibitors of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, which abolished the [Ca²⁺]_i-lowering effect of glucose on isolated alpha cells. Conclusions/interpretation Beta cell factors or somatostatin do not seem to mediate glucose inhibition of glucagon secretion. We instead propose that glucose has a direct inhibitory effect on mouse alpha cells by suppressing a depolarising Ca²⁺ store-operated current.     

Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.     

Comparison of the effects of leucines,non-metabolizable leucine analogues and other insulin secretagogues on the activity of glutamate dehydrogenase

Summary Glutamate dehydrogenase (GLDH) from bovine liver was employed in a model system for testing a possible role of GLDH in insulin release. The ability of different insulin secretagogues to stimulate the activity of the diethylstilbestrol-inhibited enzyme was tested. The two insulin-releasing amino acids, L-leucine and its non-metabolizable analogue 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid [b(−)-BCH], were the best stimulators of GLDH activity. The non-secreting stereoisomers, D-leucine and b(+)-BCH, were less effective. Glucose, L-arginine and the leucine metabolite α-ketoisocaproic acid lacked significant effects on GLDH activity. Small and diverging effects were obtained with sulfonylurea compounds: whereas carbutamide caused slight stimulation, tolbutamide and glipizide had no effect, and glibenclamide was an inhibitor. The specificity of the insulin-releasing amino acids L-leucine and b(−)-BCH in stimulating GLDH activity makes it tempting to speculate about a connection between allosteric regulation of pyridine nucleotide-dependent enzymes and insulin release.     

N-Acylated Derivatives of Sulfamethoxazole and Sulfafurazole Inhibit Intracellular Growth of Chlamydia trachomatis

Antibacterial compounds with novel modes of action are needed for management of bacterial infections. Here we describe a high-content screen of 9,800 compounds identifying acylated sulfonamides as novel growth inhibitors of the sexually transmitted pathogen Chlamydia trachomatis. The effect was bactericidal and distinct from that of sulfonamide antibiotics, as para-aminobenzoic acid did not reduce efficacy. Chemical inhibitors play an important role in Chlamydia research as probes of potential targets and as drug development starting points.     

Secretory disturbance in hyperplastic parathyroid nodules of uremic hyperparathyroidism: Implication for parathyroid autotransplantation

Pathophysiological characteristics of dispersed parathyroid cells were investigated in vitro with the principal aim of examining dominating hyperplastic parathyroid nodules in uremia. Ten large nodules were macroscopically dissected from the glands of 8 hypercalcemic patients with uremic hyperparathyroidism (HPT). Histologic examination revealed that the remaining parts of these glands were composed of a diffuse or slightly nodular parathyroid hyperplasia. Parathyroid hormone (PTH) concentrations were estimated with a mid-C regional immunoassay and the calcium indicator quin-2 was utilized to measure cytoplasmic calcium concentrations (Ca²⁺ _i) of the parathyroid cells. Ambient calcium control of PTH release and Ca²⁺ _i was significantly more abnormal in cells from the nodules while the less nodular tissue demonstrated a regulation similar to normal bovine parathyroid cells. Since an aberrant secretory regulation of the parathyroid tissue has been shown to greatly influence serum calcium concentrations in HPT, the findings substantiate a functional importance of hyperplastic parathyroid nodules in uremia. The results imply that parathyroid autotransplants should be excised from essentially nonnodular parts of the parathyroid glands since this may reduce the incidence of graft-dependent recurrences after total parathyroidectomy in uremic HPT.

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Resumen Las características patofisiológicas de células paratiroideas dispersas (preparadas mediante digestión con colagenasa) fueron investigadas in vitro con el propósito principal de analizar los nódulos hiperplásicos paratiroideos dominantes que se observan en la uremia. Diez nódulos grandes fueron disecados macroscópicamente de las glándulas de 8 pacientes hipercalcémicos con hiperparatiroidismo urémico (HPT). El examen histológico reveló que los remanentes de estas glándulas estaban compuestos por una hiperplasia paratiroidea difusa o ligeramente nodular. Las concentraciones de hormona paratiroidea (PTH) fueron determinadas por inmunoanálisis de la región media C de la molécula y se utilizó el indicador de calcio quin-2 para medir las concentraciones citoplásmicas de calcio (Ca²⁺ _i) de las células paratiroideas. El control de la secreción de PTH por el calcio ambiental y el Ca²⁺ _i aparecieron significativamente más anormales en las células de los nódulos, en tan to que el tejido menos nodular demostró una regulación similar a la de las células paratiroideas bovinas normales. Puesto que ha sido demostrado que una regulación secretoria aberrante del tejido paratiroideo influye grandemente sobre las concentraciones séricas de calcio en el HPT, esto hallazgos verifican la importancia funcional de los nódulos hiperplásicos en la uremia. Los resultados implican que los autotrasplantes paratiroideos deben ser tmnados de las porciones esencialmente no nodulares de las glándulas paratiroides ya que ello puede disminuir la incidencia de recurrencias a partir del trasplante después de paratiroidectomía total en el HPT urémico.

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Résumé Les caractéristiques physiopathologiques des cellules parathyroïdiennes dispersées ont été évaluées in vitro dans le but d'examiner les nodules d'hyperplasie parathyroïdiens de l'hyperazotémie. Dix nodules de volume important ont été disséqués des parathyroïdes chez 8 patients présentant une hyperparathyroïdie urémique (HPT). L'examen histologique a révélé que les portions restantes étaient composées d'hyperplasie parathyroïdienne peu nodulaire ou diffuse. Les concentrations en parathormones (PTH) ont été déterminées par la méthode immunologique C régional moyen; la concentration cytoplasmique en calcium (Ca²⁺ _i) (ionisé) a été mesurée par l'indicateur quin-2. Le contrôle de liberation de PTH par le calcium ambiant Ca²⁺ _i) étaient significativement perturbé dans les cellules provenant des nodules comparés à ceux des tissus peu nodulaires proches de ceux des cellules parathyroïdiennes bovines normales. Puisqu'on a démontré que la régulation sécrétrice aberrante des tissus parathyroïdiens influençait les concentrations en calcium dans HPT, ces données confirment l'importance fonctionnelle des nodules d'hyperplasie dans l'hypeazotémie. Ces résultats impliquent qu'on doit exciser et n'utiliser que les tissus non nodulaires comme implant parathyroïdien, réduisant peut-être ainsi la fréquence de récidives dépendantes du greffon après parathyroïdectomie totale pour HPT urémique.

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Presented at the International Association of Endocrine Surgeons in Sydney, Australia, September, 1987.

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Supported by the Swedish Medical Research Council, and Förenade Liv Mutual Group Life Insurance Company, Sweden.