RESUMO
The actions of extracellular Ca(2+) in regulating parathyroid gland and kidney functions are mediated by the extracellular calcium receptor (CaR), a G protein-coupled receptor. The CaR is one of the essential molecules maintaining systemic Ca(2+) homeostasis and is a molecular target for drugs useful in treating bone and mineral disorders. Ligands that activate the CaR are termed calcimimetics and are classified as either agonists (type I) or positive allosteric modulators (type II); calcimimetics inhibit the secretion of parathyroid hormone (PTH). Cinacalcet is a type II calcimimetic that is used to treat secondary hyperparathyroidism in patients receiving dialysis and to treat hypercalcemia in some forms of primary hyperparathyroidism. The use of cinacalcet among patients with secondary hyperparathyroidism who are managed with dialysis effectively lowers circulating PTH levels, reduces serum phosphorus and FGF23 concentrations, improves bone histopathology, and may diminish skeletal fracture rates and the need for parathyroidectomy. A second generation type II calcimimetic (AMG 416) is currently under regulatory review. Calcilytics are CaR antagonists that stimulate the secretion of PTH. Several calcilytic compounds have been evaluated as orally active anabolic therapies for postmenopausal osteoporosis but clinical development of all of them has been abandoned because they lacked clinical efficacy. Calcilytics might be repurposed for new indications like autosomal dominant hypocalcemia or other disorders beyond those involving systemic Ca(2+) homeostasis.
Assuntos
Calcimiméticos/uso terapêutico , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/antagonistas & inibidores , Hipercalcemia/tratamento farmacológico , Hiperparatireoidismo/tratamento farmacológicoRESUMO
The extracellular calcium receptor (CaR) is a Family C G protein-coupled receptor that controls systemic Ca2+ homeostasis, largely by regulating the secretion of parathyroid hormone (PTH). Ligands that activate the CaR have been termed calcimimetics and are classified as either Type I (agonists) or Type II (allosteric activators) and effectively inhibit the secretion of PTH. CaR antagonists have been termed calcilytics and all act allosterically to stimulate secretion of PTH. The calcimimetic cinacalcet has been approved for treating parathyroid cancer and secondary hyperparathyroidism in patients on renal replacement therapy. Cinacalcet was the first allosteric modulator of a G proteincoupled receptor to achieve regulatory approval. This review will focus on the technologies used to discover and develop allosterically acting calcimimetics and calcilytics as novel therapies for bone and mineral-related disorders.
Assuntos
Doenças Ósseas/tratamento farmacológico , Receptores de Detecção de Cálcio/metabolismo , Regulação Alostérica , Animais , Calcimiméticos/farmacologia , Calcimiméticos/uso terapêutico , Modelos Animais de Doenças , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/antagonistas & inibidoresRESUMO
Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Doenças Ósseas Metabólicas/tratamento farmacológico , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Hormônio Paratireóideo/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/fisiopatologia , Divisão Celular/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Glândulas Paratireoides/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Calcimimetics are ligands that activate the calcium receptor. Some are small molecules and of these, the most extensively studied are phenylalkylamines like cinacalcet. This compound is a positive allosteric modulator that selectively targets the parathyroid calcium receptor to inhibit the secretion of parathyroid hormone. Cinacalcet is the first calcimimetic compound to attain regulatory approval for the treatment of hyperparathyroidism resulting from end-stage renal disease. The discovery of calcimimetics and the receptor they act on are considered with the intent of extracting lessons relevant to medical research and the discovery of new drugs.
Assuntos
Cálcio/agonistas , Cálcio/fisiologia , Animais , Clonagem Molecular , Humanos , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/fisiologia , Reprodutibilidade dos TestesRESUMO
The parathyroid polyhormone hypothesis holds that peptides derived from the metabolism of parathyroid hormone (PTH) (so-called C-terminal fragments) are themselves biologically active and that their effects are mediated by a novel 'C-terminal receptor.' The evidence supporting these assertions is extensive but remains inconclusive. This Commentary focuses on in vivo pharmacology studies that provide information relevant to understanding the physiological significance of C-terminal fragments. The more recent studies of this sort provide compelling evidence that the bioactivity of C-terminal fragments is likely to become physiologically relevant in settings of secondary hyperparathyroidism. In this condition, circulating levels of C-terminal fragments greatly exceed those of PTH. There is convincing evidence that the hypocalcemic effect of C-terminal fragments results from direct actions on the skeleton that inhibit bone resorption. On the other hand, there are few if any results of in vivo studies suggesting a role for C-terminal fragments in more physiological settings, at least when parameters associated with systemic calcium homeostasis are assessed.
Assuntos
Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Hormônios Paratireóideos/metabolismo , Animais , Reabsorção Óssea/metabolismo , Humanos , Hiperparatireoidismo Secundário/metabolismo , Hiperparatireoidismo Secundário/patologia , Hiperplasia/metabolismo , Hiperplasia/patologia , Glândulas Paratireoides/patologiaRESUMO
The cell surface calcium receptor (Ca2+ receptor) is a particularly difficult receptor to study because its primary physiological ligand, Ca2+, affects numerous biological processes both within and outside of cells. Because of this, distinguishing effects of extracellular Ca2+ mediated by the Ca2+ receptor from those mediated by other mechanisms is challenging. Certain pharmacological approaches, however, when combined with appropriate experimental designs, can be used to more confidently identify cellular responses regulated by the Ca2+ receptor and select those that might be targeted therapeutically. The Ca2+ receptor on parathyroid cells, because it is the primary mechanism regulating secretion of parathyroid hormone (PTH), is one such target. Calcimimetic compounds, which active this Ca2+ receptor and lower circulating levels of PTH, have been developed for treating hyperparathyroidism. The converse pharmaceutical approach, involving calcilytic compounds that block parathyroid cell Ca2+ receptors and stimulate PTH secretion thereby providing an anabolic therapy for osteoporosis, still awaits clinical validation. Although Ca2+ receptors are expressed throughout the body and in many tissues that are not intimately involved in systemic Ca2+ homeostasis, their physiological and/or pathological significance remains speculative and their value as therapeutic targets is unknown.
Assuntos
Cálcio/metabolismo , Receptores de Detecção de Cálcio/fisiologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Canais de Cálcio/fisiologia , Cinacalcete , Fendilina/análogos & derivados , Fendilina/química , Fendilina/farmacologia , Humanos , Hiperparatireoidismo/tratamento farmacológico , Naftalenos/química , Naftalenos/farmacologia , Osteoporose/tratamento farmacológico , Glândulas Paratireoides/efeitos dos fármacos , Glândulas Paratireoides/metabolismo , Fenetilaminas , Propilaminas , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/antagonistas & inibidores , EstereoisomerismoRESUMO
The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in melanotrophs, the characteristic endocrine cells of the pars intermedia of the rat pituitary gland, using the fluorescent Ca indicator fura-2. The resting [Ca2+]i was 211 +/- 8 nM and was little affected by tetrodotoxin (TTX; 5 or 10 microM), which inhibits the spontaneous action potentials that occur in these cells. Removal of extracellular Ca2+ (by chelation with EGTA) or addition of the Ca channel blocker nimodipine (1 microM) produced a rapid fall in [Ca2+]i, which occurred whether TTX was present or not. Excess K+ (60 mM), veratridine (10 or 100 microM) and BAY K 8644 (1 microM) each caused a rapid rise in [Ca2+]i, which was blocked or truncated by EGTA or nimodipine. TTX blocked or truncated the increases in [Ca2+]i induced by veratridine, but not those induced by either excess K+ or BAY K 8644. The results show that manipulations that increase or decrease hormone output increase or decrease [Ca2+]i. Furthermore, the resting [Ca2+]i appears to depend importantly on Ca influx, since it is rapidly and markedly reduced by removal of extracellular Ca2+ or addition of a Ca channel blocker.
Assuntos
Cálcio/metabolismo , Canais Iônicos/fisiologia , Hipófise/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Citosol/metabolismo , Ácido Egtázico/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Nimodipina/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/ultraestrutura , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Tetrodotoxina/farmacologia , Veratridina/farmacologiaRESUMO
The level of extracellular ionized calcium ([Ca2+]o) is the primary physiological regulator of PTH secretion. Complementary DNAs encoding the calcium receptor (CaR) protein that mediates this response have been cloned from bovine and human parathyroid glands. This protein is a seven-transmembrane, G-protein-coupled receptor linked to the mobilization of intracellular Ca2+ in response to increases in [Ca2+]o. More recently, a rat kidney CaR has been cloned and shown to be 92% identical at the amino acid level to the bovine parathyroid CaR. Homologous or heterologous regulation of the expression and/or function of a variety of G-protein-coupled receptors has been documented in numerous cell types. Therefore, we determined whether [Ca2+]o and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], major regulators of PTH synthesis and secretion, affect CaR gene expression in parathyroid gland and kidney in rats. CaR messenger RNA (mRNA) levels were quantified in pairs of parathyroid glands and single kidneys from individual animals using a solution hybridization assay. The effects of Ca2+ and 1,25-(OH)2D3 on CaR gene expression were assessed independently in vitamin D-deficient (-D) rats. A wide range of plasma Ca2+ levels (0.7-1.9 mM) was produced by supplementing -D diets with varying amounts of calcium and by infusing CaCl2 i.v. for 7 days using osmotic minipumps. There was no correlation between plasma Ca2+ levels and steady state CaR mRNA levels in parathyroid gland (r = -0.18) or kidney (r = 0.25). In another group of -D rats, 1,25-(OH)2D3 was infused sc at 25 and 275 ng/kg.day for 10-12 days. Dietary calcium was adjusted to maintain normocalcemia in some of the groups. No effect of 1,25-(OH)2D3 administration on CaR mRNA levels occurred in parathyroid glands or kidney regardless of the resultant plasma Ca2+ or 1,25-(OH)2D3 levels. In conclusion, neither parathyroid gland nor kidney CaR mRNA levels are regulated by plasma Ca2+ and 1,25-(OH)2D3 levels in the experimental models examined here.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/administração & dosagem , Colecalciferol/deficiência , Rim/metabolismo , Glândulas Paratireoides/metabolismo , Deficiência de Vitamina D/metabolismo , Animais , Calcitriol/sangue , Cálcio/sangue , Cálcio da Dieta , Bovinos , Colecalciferol/administração & dosagem , Colecalciferol/sangue , Dieta , Humanos , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The concentration of intracellular Ca2+ [( Ca2+]i) was measured in dissociated bovine parathyroid cells loaded with quin-2 or fura-2. In quin-2-loaded cells, increases in the concentration of extracellular Ca2+ elicited slow, monophasic increases in [Ca2+]i, whereas in fura-2-loaded cells, extracellular Ca2+ evoked rapid, transient increases which were followed by lower, yet sustained increases in [Ca2+]i. Cytosolic Ca2+ transients arose from the mobilization of cellular Ca2+ and could be evoked by a variety of divalent cations. Transient, but not sustained increases in [Ca2+]i were associated with an inhibition of hormone secretion. Secretion was still inhibited, however, when cytosolic Ca2+ transients were blocked by buffering with quin-2, suggesting that changes in [Ca2+]i might not be the essential factor regulating secretion in parathyroid cells.
Assuntos
Cálcio/fisiologia , Glândulas Paratireoides/metabolismo , Aminoquinolinas , Animais , Benzofuranos , Bovinos , Citosol/fisiologia , Fura-2 , Lantânio/farmacologia , Magnésio/farmacologia , Taxa Secretória/efeitos dos fármacosRESUMO
Various studies suggest the existence of a plasma membrane receptor on parathyroid cells that senses changes in the concentration of extracellular Ca2+. To test this hypothesis, Xenopus laevis oocytes were injected with poly(A)(+)-enriched mRNA from bovine parathyroid cells and examined for their ability to respond to increases in the concentration of extracellular Ca2+ or other polycations. Cytosolic Ca2+ concentrations were measured indirectly by recording Cl- currents through the endogenous, cytosolic Ca(2+)-activated Cl- channel. Increasing the concentration of extracellular Ca2+ (from 0.7 to 5 mM) or Mg2+ (from 0.8 to 10 mM) elicited oscillatory increases in the Cl- current. Responses to either divalent cation were not observed in oocytes injected with water or with mRNA prepared from HL-60 cells or rat liver. Responses elicited by extracellular Mg2+ persisted when extracellular Ca2+ was reduced to low micromolar levels. La3+, Gd3+, or neomycin B also evoked oscillatory increases in the Cl- current in oocytes under conditions of low extracellular Ca2+ levels. These extracellular polycations all cause the mobilization of intracellular Ca2+ in oocytes injected with parathyroid cell mRNA like they do in intact parathyroid cells. The injection of parathyroid cell mRNA thus confers on oocytes the ability to detect and respond to changes in the concentration of extracellular polycations. The data provide compelling evidence for the existence of a cell surface Ca2+ receptor protein(s) on parathyroid cells that regulates cellular function.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Glândulas Paratireoides/metabolismo , Animais , Cátions/farmacologia , Bovinos , Células Cultivadas , Clonagem Molecular , Feminino , Oócitos/metabolismo , Glândulas Paratireoides/citologia , Xenopus laevisRESUMO
Parathyroid hormone (PTH) is the key endocrine factor regulating systemic Ca(2+) homeostasis. Elevated levels of circulating PTH increase bone turnover and, depending on the duration of elevation, will result in net anabolic or catabolic effects on the skeleton. Secretion of PTH from the parathyroid glands is regulated by small changes in circulating levels of Ca(2+) which are detected by a Ca(2+) receptor on the surface of parathyroid cells. This G protein-coupled receptor is the primary molecular entity used by parathyroid cells to regulate secretion of PTH. As such, the Ca(2+) receptor is a unique molecular target for new drugs capable of increasing or decreasing circulating levels of PTH. Compounds which activate the Ca(2+) receptor are termed calcimimetics and they inhibit the secretion of PTH; a calcimimetic compound is in late stage clinical trials for the treatment of both primary and secondary hyperparathyroidism. Conversely, calcilytic compounds, which are Ca(2+) receptor antagonists, stimulate secretion of PTH; a calcilytic compound is in early clinical development for the treatment of osteoporosis.
Assuntos
Cálcio/metabolismo , Hiperparatireoidismo/tratamento farmacológico , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Animais , Cálcio/química , Ensaios Clínicos como Assunto , Humanos , Hiperparatireoidismo/metabolismo , Hiperparatireoidismo/fisiopatologia , Hiperparatireoidismo Secundário/tratamento farmacológico , Hiperplasia , Mimetismo Molecular , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Glândulas Paratireoides/patologia , Hormônio Paratireóideo/antagonistas & inibidores , Receptores de Detecção de Cálcio , Receptores de Superfície Celular/metabolismoRESUMO
Parathyroid hormone (PTH) administration increases bone mass in normal and osteopenic animals. However, this treatment currently requires the daily injection of large amounts of PTH, and the relationship of these doses to plasma levels of PTH that are achievable physiologically is unknown. We determined in ovariectomized (ovx) rats: 1) the plasma PTH levels that occur after the subcutaneous injection of graded doses of rat PTH, 2) whether similar PTH levels can be achieved by stimulation of endogenous PTH secretion, and 3) whether a plasma PTH profile that is achievable physiologically is anabolic on bone. Injection of 1, 5, or 25 micrograms/kg rat PTH-(1-34) increased plasma PTH by 46, 164, or 520 pg/mL, respectively, above basal levels within 60 min. Infusion of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid for 2 h reduced plasma Ca2+ by 0.36 mmol/L and produced a total plasma PTH response (area under the plasma PTH curve) similar to that with the 5 micrograms/kg rat PTH injection. Then, 1, 5, or 25 micrograms/kg doses of rat PTH-(1-34) were injected subcutaneously daily for 28 days in 19-week-old rats that were ovx 7 weeks earlier. The 5 and 25 micrograms/kg doses significantly increased bone mineral density in the distal femur and trabecular bone area and average trabecular thickness in the proximal tibia. All doses of PTH significantly increased indices of trabecular connectivity and cancellous bone formation, including double-labeled surface, mineralizing surface, and surface-referent bone formation rate. In conclusion, anabolic effects on bone can be achieved with a plasma PTH profile similar to that attained following stimulation of the parathyroid gland by induced hypocalcemia. These data suggest that agents that transiently increase endogenous PTH secretion may represent a novel means to promote anabolic effects in skeletal tissues.
Assuntos
Densidade Óssea/efeitos dos fármacos , Desenvolvimento Ósseo/efeitos dos fármacos , Ácido Egtázico , Hormônio Paratireóideo/sangue , Teriparatida/farmacologia , Absorciometria de Fóton , Animais , Cálcio/sangue , Feminino , Infusões Intravenosas , Injeções Subcutâneas , Masculino , Ovariectomia , Hormônio Paratireóideo/administração & dosagem , Hormônio Paratireóideo/metabolismo , Ratos , Ratos Sprague-Dawley , Teriparatida/administração & dosagem , Teriparatida/farmacocinética , Tíbia/efeitos dos fármacos , Tíbia/metabolismoRESUMO
The calcium receptor agonist (calcimimetic) compound NPS R-568 causes rapid decreases in circulating levels of parathyroid hormone (PTH) in rats and humans. We hypothesized that daily intermittent decreases in serum PTH levels may have different effects on bone than do chronically sustained decreases. To test this hypothesis, we compared two NPS R-568 dosing regimens in rats with chronic renal insufficiency induced by two intravenous injections of adriamycin. Fourteen weeks after the second adriamycin injection, creatinine clearance was reduced by 52%, PTH levels were elevated approximately 2.5-fold, and serum 25(OH)D3 and 1,25(OH)2D3 levels were reduced substantially. Treatment by daily per os gavage, which decreased PTH levels intermittently, or continuous subcutaneous infusion, which resulted in a sustained suppression of serum PTH levels, then began for 8 weeks. Despite the hyperparathyroidism, the adriamycin-injected rats developed a low-turnover bone lesion with osteomalacia (fourfold increase in osteoid volume in the proximal tibial metaphysis) and osteopenia (67% decrease in cancellous bone volume and an 18% reduction in bone mineral density at the distal femur). Daily administered (but not infused) NPS R-568 significantly increased cancellous bone volume solely by normalizing trabecular thickness, and increased femoral bone mineral density by 14%. These results indicate that daily intermittent, but not sustained, decreases in PTH levels have an "anabolic-like" effect on bones with a low-turnover lesion in this animal model of chronic renal insufficiency.
Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/patologia , Osteomalacia/metabolismo , Osteomalacia/patologia , Hormônio Paratireóideo/sangue , Uremia/metabolismo , Uremia/patologia , Compostos de Anilina/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Cálcio/agonistas , Cálcio/sangue , Distúrbio Mineral e Ósseo na Doença Renal Crônica/etiologia , Distúrbio Mineral e Ósseo na Doença Renal Crônica/metabolismo , Distúrbio Mineral e Ósseo na Doença Renal Crônica/patologia , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Humanos , Hiperparatireoidismo Secundário/etiologia , Hiperparatireoidismo Secundário/metabolismo , Hiperparatireoidismo Secundário/patologia , Masculino , Osteomalacia/etiologia , Fenetilaminas , Fosfatos/sangue , Propilaminas , Ratos , Ratos Sprague-Dawley , Uremia/etiologiaRESUMO
The Ca(2+) receptor on the surface of parathyroid cells is the primary molecular entity regulating secretion of parathyroid hormone (PTH). Because of this, it is a particularly appealing target for new drugs intended to increase or decrease circulating levels of PTH. Calcilytic compounds are Ca(2+) receptor antagonists which increase the secretion of PTH. The first reported calcilytic compound was NPS 2143, an orally active molecule which elicits rapid, 3- to 4-fold increases in circulating levels of PTH. These rapid changes in plasma PTH levels are sufficient to increase bone turnover in ovariectomized, osteopenic rats. When administered together with an antiresorptive agent (estradiol), NPS 2143 causes an increase in trabecular bone volume and bone mineral density in osteopenic rats. The magnitude of these changes are far in excess of those caused by estradiol alone and are comparable with those achieved by daily administration of PTH or a peptide analog. These anabolic effects of NPS 2143 on bone are not associated with hyperplasia of the parathyroid glands. Calcilytic compounds can increase endogenous levels of circulating PTH to an extent that stimulates new bone formation. Such compounds could replace the use of exogenous PTH or its peptide fragments in treating osteoporosis.
Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Animais , Feminino , Naftalenos/farmacologia , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/metabolismo , RatosRESUMO
We have examined the effects of a number of excitatory amino acid antagonists on transmission at the cochlear nerve-nucleus magnocellularis synapse in the chicken. Using an in vitro preparation and bath application of drugs, we studied the effects of kynurenic acid and several related substances, streptomycin and a selective N-methyl-D-aspartate receptor antagonist, DL-alpha-aminosuberate. The last compound had no effect on evoked transmission. Of the various kynurenic acid-related compounds tested, only kynurenic and dipicolinic acid selectively altered responses in nucleus magnocellularis. Quinolinic acid, a kynurenic acid analogue that is structurally akin to dipicolinic acid but which acts selectively at N-methyl-D-aspartate receptors, was without effect. The effect of kynurenic acid was solely inhibitory, completely blocking postsynaptic responses with a potency dependent on the frequency of nerve stimulation. No such frequency dependence was seen with dipicolinic acid although this compound also completely suppressed evoked responses. In addition dipicolinic acid potentiated postsynaptic responses at concentrations only slightly lower than those causing inhibition. Streptomycin inhibited responses in nucleus magnocellularis but this effect seems to result partially from the ability of the drug to inhibit presynaptic calcium influx. Our finding that selective antagonists of N-methyl-D-aspartate receptors were ineffective while antagonists of both receptor types, such as kynurenic and dipicolinic acids, inhibited evoked responses reinforces the conclusion that postsynaptic receptors mediating transmission at this synapse are of the non-N-methyl-D-aspartate type [Nemeth et al. (1983) Neurosci. Lett. 40, 39-44].(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Nervo Coclear/efeitos dos fármacos , Ácido Cinurênico/farmacologia , Neurônios/fisiologia , Ácidos Picolínicos/farmacologia , Receptores de Neurotransmissores/fisiologia , Estreptomicina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Cálcio/farmacologia , Galinhas , Nervo Coclear/citologia , Nervo Coclear/fisiologia , Técnicas de Cultura , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Ácido Cinurênico/análogos & derivados , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato , Receptores de Neurotransmissores/classificação , Receptores de Neurotransmissores/efeitos dos fármacos , Estreptomicina/metabolismo , Fatores de TempoRESUMO
It is not clear whether hypercalcemia and hyperparathyroidism associated with lithium therapy are the result of an unmasking of preexisting disease or a direct effect of lithium on the parathyroid glands. To investigate this phenomenon, parathyroid hormone (PTH) secretion and cytosolic calcium concentrations [( Ca]i) were measured in normal and lithium-treated dispersed bovine parathyroid cells grown in tissue culture and incubated with varying concentrations of extracellular calcium [( Ca]e) (0.5 to 2.5 mmol/L). Results indicate that lithium has two effects on parathyroid secretory response: (1) a decrease in low calcium-stimulated PTH release and (2) a potentiation of PTH release at physiologic concentrations of extracellular calcium. [Ca]i was assessed by use of fura-2, a calcium-sensitive fluorescent indicator. Resting [Ca]i levels were unaffected by lithium (103 +/- 13 nmol/L in controls vs 101 +/- 5 nmol/l in lithium-treated cells, mean +/- SE). Subsequent increases in [Ca]i in response to increases in [Ca]e were significantly less in lithium-treated cells, with no difference at maximal [Ca]e. Increases in [Ca]i in response to a submaximal concentration of extracellular magnesium were also blunted in cells pretreated with lithium. In conclusion, our data suggest that, at physiologic calcium concentrations, lithium decreases parathyroid cell sensitivity to changes in [Ca]e, reducing [Ca]i levels and increasing PTH secretory response.
Assuntos
Lítio/farmacologia , Glândulas Paratireoides/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bovinos , Técnicas de Cultura , Glândulas Paratireoides/citologia , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/metabolismoRESUMO
The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.
Assuntos
Cálcio/metabolismo , Cerebelo/metabolismo , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Citosol/metabolismo , Ácido Egtázico/farmacologia , Fura-2 , Glicina/farmacologia , Cinética , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
ETYA (5,8,11,14-eicosatetraynoic acid; 50-100 microM), which inhibits both cyclo-oxygenase and lipoxidase, inhibited histamine release evoked by secretagogues dependent on extracellular calcium (antigen, dextran, and concanavalin A) but failed to inhibit secretion elicited by secretagogues capable of mobilizing calcium from intracellular sites (48/80, polymyxin B, protamine sulfate and poly-L-lysine). Responses to these latter secretagogues were inhibited only by higher concentrations of ETYA (100-200 microM) that were cytotoxic. Secretion evoked by the calcium ionophore A23187 (0.1 microgram/ml) was inhibited at much lower concentrations of ETYA (1-10 microM) but this inhibition could not be overcome by increasing the concentration of calcium. Responses to higher concentrations of ionophore were not inhibited by ETYA except in amounts affecting cell viability. Like ETYA, each of several fatty acids, including arachidonic acid, were inhibitory towards histamine release evoked by A23187 or 48/80. The results indicate that EYTA acts at some early stage of stimulus-secretion coupling rather than on the final, common, calcium-activated steps of exocytosis. Moreover, this action may be unrelated to inhibition of lipoxidase or cyclooxygenase.
Assuntos
Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Mastócitos/efeitos dos fármacos , Animais , Calcimicina/farmacologia , Concanavalina A/farmacologia , Liberação de Histamina/efeitos dos fármacos , Técnicas In Vitro , Masculino , Mastócitos/fisiologia , Ratos , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
The lipoxygenase inhibitors HTYA (5,8,11,14-henicosatetraynoic acid, 25-150 microM) and ITYA (4,7,10,13-icosatetraynoic acid, 25-75 microM), inhibited histamine secretion from rat mast cells evoked by concanavalin A but that elicited by compound 48/80 or polymyxin B. Arachidonic acid (100 microM) did not inhibit concanavalin A-induced histamine secretion. The results are consistent with the notion that lipoxygenase inhibitors affect an early stage of stimulus-secretion coupling in the mast cell, peculiar to antibody-directed secretagogues, perhaps that involving calcium influx.
Assuntos
Exocitose/efeitos dos fármacos , Inibidores de Lipoxigenase , Mastócitos/efeitos dos fármacos , Ácido 5,8,11,14-Eicosatetrainoico/análogos & derivados , Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Animais , Concanavalina A/farmacologia , Liberação de Histamina/efeitos dos fármacos , Masculino , Polimixina B/farmacologia , Ratos , Ratos Endogâmicos , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Previous studies using various excitatory amino acid antagonists have shown that synaptic transmission between the auditory nerve and the cochlear nucleus of chickens (nuc. magnocellularis; NM) is mediated by non-N-methyl-D-aspartate (non-NMDA) receptors. In the present study we have attempted to define the subclass of non-NMDA receptor in the NM by examining the effects of various excitatory amino acid agonists on synaptically evoked field potentials in an in vitro preparation of the chicken brain stem. Both quisqualate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), whose actions operationally define the quisqualate receptor class, caused variable and weak depression of evoked responses in the NM, as did L-glutamate. Kainic acid, on the other hand, completely blocked postsynaptic responses at micromolar concentrations. We conclude that kainate-preferring non-NMDA receptors play a predominant role in mediating transmission in the NM.