Effects of calcium and cyclic nucleotides on rat calcitonin and parathyroid hormone secretion.

Recently we developed a system for studying concurrent secretion of calcitonin (CT)and parathyroid hormone(PTH)in vitro from single rat thyroparathyroid gland complexes. In the present study, mechanisms involved in secretion of CT and PTH were explored by altering the medium [Ca ] and by using the Ca antagonist, verapamil. We also re-examined the idea that cyclic nucleotides may help regulate secretion of these hormones and attempted to determine if effects of cyclic nucleotides might be altered by changes in medium [Ca]. Thyroparathyroid glands from 8-day-old rats were incubated in serum-free medium for 8h, and CT and PTH levels in the medium were measured by radioimmunoassays. We show for the first time that: (1) although low [Ca] is well known to promote PTH release, some extracellular Ca is needed for PTH secretion to occur at a maximal rate; (2) inhibition of Ca entry into cells with verapamil mimics the effects of low medium Ca on both CT and PTH release; and (3) cyclic nucleotides may exert their effects on secretion of CT and PTH at least in part via effects on Ca entry into cells.     

Studies on the control and dynamics of thyrotropin secretion from isolated adenohypophyseal cells.

Thyrotropin (TSH) secretion from isolated anterior pituitary cells has been studied using the technique of cell column perifusion. The consistency in secretory rate and temporal profiles of TSH output in response to stimulation illustrated that the system is suitable for studying the kinetics of stimulation and inhibition of secretion. During perfusion TSH release was stimulated in response to a variety of secretogogues, namely TRH, raised potassium concentrations and phosphodiesterase inhibitors. The onset and termination of the secretory responses were rapid and displayed a temporally biphasic pattern of secretion. Dose-related increases in TSH output in response to TRH and consistent responses to repetitive pulses of TRH (5.5 X 10-10 M) during a 4 h period were demonstrated. Studies on the dynamics of thyroid hormone feedback on TRH-stimulated TSH secretion indicated that inhibition was manifest within 1 h and reached a maximum after 2 1/2 h during continual exposure to thyroid hormones. Isobutylmethylxanthine (IBMX) potentiated the effect of raised K+ as well as that of TRH on TSH secretion, suggesting an as yet unidentified relationship between Ca2+ and cyclic AMP.     

TSH, theophylline and cyclic AMP: in vitro thyroid activity in aging rats.

In vitro thyroid accumulation of cyclic 3',5'-adenosine monophosphate (cyclic AMP) and release of triiodothyronine (T₃) and thyroxine (T₄) in response to TSH, theophylline or cyclic AMP treatment was assessed in 60- and 340-day-old male rats. Plasma levels of T₃ and T₄ at the time of sacrifice were determined. Mature animals exhibited significantly lower plasma T₃ and T₄ levels but slightly elevated in vitro secretory rates of T₃ and T₄. TSH stimulation elicited little effect in the mature gland in terms of cyclic AMP accumulation or thyroid hormone release. Conversely, cyclic AMP enhanced in vitro thyroid hormone release in both age groups. The data suggest an age-related alteration in thyroid responsiveness to TSH which in turn may be a function of changes in the thyroidal adenylate cyclase-cyclic AMP-phosphodiesterase system. Evidence is also presented which suggests either cyclic AMP-mediated T₄ to T₃ conversion or a differential action of the nucleotide upon T₃ as compared to T₄ synthesis.     

Thyroxine secretion by isolated hog thyroid cells: a cyclic AMP independent pathway.

The release of 131I-labeled thyroxine (T4) from isolated hog thyroid cells was increased 1.5--2-fold by thyrotropin (TSH). Dibutyryl cyclic AMP failed to reproduce this TSH action. In this in vitro system another cell activity, T4 synthesis, was stimulated in an essentially identical fashion by TSH and dibutyryl cyclic AMP (time course of action, dose-response relationship). 3-Isobutyl-1-methylxanthine (IBMX), 0.5 mM, did not alter the basal [131I]T4 release whereas it enhanced the [131I]T4 synthesis. TSH, 60 MU/ml, increased the intracellular cyclic AMP concentration 3-4-fold. Chlorpromazine (5 X 10(-4)M) abolished the TSH stimulation of cyclic AMP accumulation but did not alter the TSH-induced increase in [131I]T4 secretion. It is concluded that the TSH action on [131I]T4 secretion by isolated thyroid cells is not mediated by the adenylate cyclase-cylic AMP system.     

Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line

We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 microM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating 'uncoupling' of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of the adenylate cyclase by epinephrine (10(-6) M-10(-4) M in the presence of 10(-4) M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.     

Further characterization of the iodide inhibitory effect on the cyclic AMP system in dog thyroid slices

Iodide inhibits cyclic AMP accumulation in the thyroid by a process which is prevented by inhibition of iodide uptake and of thyroid peroxidase. By a similar process, it also exerts other independent effects such as the enhancement of iodinated protein release. Iodide inhibited the stimulation of adenylate cyclase by prostaglandin E₁, cholera toxin and forskolin. The action of iodide was not relieved by phosphodiesterase inhibitors and was not additive with the effect of norepinephrine or adenosine. Iodide did not decrease the cellular level of ATP. The data are compatible with an inhibition of adenylate cyclase beyond the level of the receptor, presumably at the level of the catalytic unit or its interaction with the positive transducing unit N_s. The effect of iodide required TSH for its expression but not for its installation. It was decreased under all conditions in which iodide organification was decreased: decreased iodide or increased methimazole concentration, absence of calcium in the medium, etc. However, the relation between iodide binding to proteins and effect was not linear. The effect was not relieved by washing in the absence of iodide and in the presence of perchlorate, but it was partly reversible in the presence of methimazole propylthiouracyl or thiourea. It was not relieved by cooling to 20°C and cytochalasin b, which block stimulated thyroglobulin hydrolysis and iodothyronine release, nor by actinomycin D, cycloheximide, puromycin, mepacrine or indomethacin. The data suggest that iodide binds to a saturable cell component by a reaction which is reversible only in the presence of thiol-containing drugs.     

Studies of the role of opioids in control of human pancreatic secretion

Opioids, both endogenous and exogenous, have been shown to exert a modulating influence on many gastrointestinal functions. Animal studies suggest an inhibitory role for opioids in the control of pancreatic secretion, which may be exerted through their inhibitory effect on acetylcholine release from intramural neurons. We studied pancreatic secretion in six healthy male volunteers (mean age 42 years) by means of duodenal aspiration using PEG-4000 as a recovery marker. Pancreatic secretion was stimulated with duodenal perfusion of mannitol (510 mosmol/kg) and an equimolar solution (0.3 M/liter) ofl-phenylalanine andl-tryptophan and also with an intravenous bolus of secretin and cholecystokinin (each 1 CU/kg). Each subject underwent six studies on separate days with intravenous infusion of saline, naloxone 40 g/kg/hr, morphine sulfate 40 g/kg/hr, morphine and naloxone combined, naloxone and atropine 13 g/kg/hr, and morphine and bethanechol 16 g/kg/hr. Naloxone caused a significant increase in amylase outputs during the basal period and in response to all stimuli, without affecting bicarbonate outputs. This effect was blocked by atropine. Morphine caused significant reductions in both amylase and bicarbonate outputs with all stimuli. This was accompanied by a reduction in amylase concentrations but not in those of bicarbonate. Bethanechol counteracted these effects of morphine. The combination of morphine and naloxone had no significant effect on pancreatic secretion. Changes in pancreatic polypeptide levels did not explain the observations. In conclusion, these studies suggest that endogenous opioids exert a modulating influence on human pancreatic secretion. Morphine has a significant inhibitory effect on enzyme output as in animal studies. These effects may be mediated through an effect on acetylcholine release from nerve terminals and an interaction with cholecystokinin.     

Lanthanum inhibition of calcitonin secretion and calcium uptake in porcine thyroid slices

The influence of lanthanum on theophylline-induced calcitonin secretion and ⁴⁵Ca uptake by slices of porcine thyroid was examined in the present study. Theophylline (4 mM) produced a significant rise in calcitonin release; however, tissue uptake of ⁴⁵Ca was not affected. In the presence of 10 mM lanthanum ⁴⁵Ca uptake was reduced in both the control and theophylline-treated tissue and theophylline-stimulated calcitonin secretion was abolished. It is concluded that the calcium ion is necessary for theophylline-mediated calcitonin secretion.     

Biliary calcium and bile acid secretion in intact and TPTX rats with varying plasma calcium concentration

Investigations of the effects of plasma calcium concentration on the relationship between biliary secretion of bile acid and calcium were performed in normocalcemic, calcium gluconate-induced hypercalcemie, thyroparathyroidectomy-induced hypocalcemic (TPTX) rats, and TPTX rats that received calcium gluconate to maintain normocalcemia. Studies were done at normal bile flow and at sodium taurocholate-stimulated bile flow. The results showed that biliary calcium secretion, which could occur in the absence of parathyroid hormone and calcitonin, was dependent mainly on plasma calcium concentration and was only partly influenced by bile acid secretion. Concerning the route of biliary calcium secretion, 80% was by the transcellular pathway and 20% was by the paracellular pathway. During theophylline-stimulated bile-acid-independent bile flow, the increase in bile-acid-independent calcium was found to be secreted by both pathways.This work was supported in part by a research grant from Mahidol University.     

Stimulation of luteinising hormone release by luteinising hormone-releasing hormone in the porcine anterior pituitary: The role of cyclic AMP

Cells dissociated from porcine pituitary glands have been used to examine the relationship between cyclic AMP levels and the stimulation of luteinising hormone (LH) secretion by luteinising hormone-releasing hormone (LHRH). It has been shown that while dibutyryl cyclic AMP and theophylline have a modest and, under some conditions, an inhibitory effect on LH release, the phosphodiesterase inhibitors isobutylmethylxanthine and ICI 63197 both induce LH release and enhance LHRH-induced release. The prostaglandins E₁ and E₂ and cholera toxin, which are shown to raise cyclic AMP levels in the dissociated preparation, also induce LH release and increase LHRH-induced LH release. Finally, LHRH is shown to increase the total content of cyclic AMP in the mixed cell preparation by approximately 30% and it is concluded that, in this system, the nucleotide does play a role in coupling LHRH stimulation to LH release.     

Separation and analysis of two plasma membrane fractions from bovine thyroid which differ in TSH binding and TSH activation of adenylate cyclase

A tissue disruption technique leading to the separation of thyroid epithelial cell components from interfollicular material has been used to study the distribution and the properties of membrane adenylate cyclase originating from intraglandular thyroid and non-thyroid cells. Bovine thyroid fragments were forced through a metallic sieve. The material which filtrates was composed of open cells and cell debris (fraction A); the material remaining on the sieve contained the basal lamina and the interfollicular material as shown by photon and electron microscopic observations (fraction B). Homogenates (HA and HB) were prepared from fractions A and B and centrifuged on a 41% sucrose layer to prepare membrane fractions: MA and MB, which were tested for the presence of adenylate cyclase, TSH-responsive adenylate cyclase and 125I-labelled TSH binding activity. HA and HB contained respectively 70% and 30% of the total thyroid adenylate cyclase activity. MA and MB were similarly enriched in 5'-nucleotidase and adenylate cyclase: 8- to 10-fold as compared to the corresponding homogenates. MA and MB exhibited a marked difference in the response to TSH: TSH either alone or in the presence of Gpp(NH)p stimulated the adenylate cyclase of MA and did not have any effect on MB. Fractionation of MA by isopycnic centrifugation on Percoll gradients yielded a membrane peak exhibiting a TSH-responsive adenylate cyclase activity and a 125I-labelled TSH binding activity displaceable by an excess of unlabelled TSH. A membrane peak at the same density was obtained from MB but its adenylate cyclase did not respond to TSH and there was no specific binding of labelled TSH. Our data indicate that an important fraction of membrane adenylate cyclase of the thyroid does not seem to be coupled with TSH receptor; the major part of this fraction (MB) likely originates from intraglandular non-thyroid epithelial cells. The separation of this membrane fraction from the thyroid cell plasma membrane fraction (MA) allows to increase the response of this latter fraction to TSH.     

自主性功能性甲状腺腺瘤TSH受体基因突变研究

目的　研究TSH受体 (TSHR)基因突变在自主性功能性甲状腺腺瘤 (AFTA)发病中的作用。方法　以 14例AFTA作为研究对象 ,另 4例为毒性多结节性甲状腺肿、7例扫描呈“冷结节”的甲状腺腺瘤及AFTA周围正常甲状腺组织作为对照 ,酚 氯仿 异戊醇法提取基因组DNA ,对目的基因片段进行聚合酶链反应—单链构象多态性 (PCR SSCP)分析及DNA序列分析。结果　在 14例自主性功能性甲状腺瘤标本中 ,SSCP检测出 6例条带变异的个体 (4 3 % ) ,对其中 3例进行DNA序列测定 ,发现 1例为 6 2 0位密码子的点突变 ,苏氨酸被脯氨酸置换 (T6 2 0P ,ACC→CCC)。 2例为单碱基插入突变 ,在 1972与 1973位核苷酸之间插入了一个腺嘌呤核苷酸 (A) ,使得密码子 6 2 4位以后的氨基酸发生了移码突变。在对照组未发现TSHR基因突变。结论　TSHR基因突变可能在自主性功能性甲状腺腺瘤的发病中起重要作用。     

Effect of misoprostol on histamine-stimulated acid secretion and cyclic AMP formation in isolated canine parietal cells

Isolated canine parietal cells were used to study the ability of misoprostol to inhibit acid secretion in the presence of a number of acid secretagogues. Misoprostol inhibited histamine-stimulated acid secretion in a dose-dependent and noncompetitive manner. A concentration of 2–3×10^–9 M misoprostol inhibited maximal histamine-stimulated acid secretion by one half. Misoprostol had little to no effect on acid secretion stimulated by carbachol and dibutyryl cAMP, had no effect on the acid secretion directly stimulated by pentagastrin, and only modestly inhibited acid secretion stimulated by forskolin. Misoprostol noncompetitively inhibited cAMP formation in response to histamine, with an IC₅₀ value similar to that for the inhibition of histamine-stimulated acid secretion. These results indicate that: (1) misoprostol specifically inhibits histamine-stimulated acid secretion in parietal cells, and (2) the antisecretory action of misoprostol is closely related to the reduction of histamine-stimulated cAMP formation with the site of major action most likely in the coupling process between histamine H₂ receptor sites and histamine-sensitive adenylate cyclase.     

Difference in calcium dependency of insulin,glucagon and somatostatin secretion in response to glibenclamide in perfused rat pancreas

Summary The extracellular calcium requirements for insulin, glucagon and somatostatin release induced by 1 g/ml of glibenclamide have been compared in the perfused, isolated rat pancreas. In the absence of glucose, the drug evoked insulin release equally well at physiological (2.6 mmol/l) and low (0.25 mmol/l) levels of total calcium. In contrast, glibenclamide evoked somatostatin release at 2.6 but not at 0.25 mmol/l of calcium. At 2.6 mmol/l of calcium, glibenclamide evoked bimodal effects (stimulation followed by inhibition) on glucagon secretion. At 0.25 mmol/l of calcium, basal secretory rates of glucagon were elevated and a small stimulatory effect of glibenclamide was seen. Addition of 0.5 mmol/l of EGTA to media with low calcium concentrations uniformly abolished the A, B and D cell secretory responses to glibenclamide. The possible modulation of calcium dependency by a non-stimulatory concentration of glucose was tested by its addition at 3.3 mmol/l to the perfusion media. Glucose enhanced glibenclamide-induced insulin secretion, both at 0.25 and 2.6 mmol/l of calcium. However, at 0.25 mmol/l of calcium, the enhancing effect of glucose was more pronounced than at 2.6 mmol/l. At 2.6 mmol/l of calcium, glucose diminished the somatostatin and abolished the glucagon response to glibenclamide. At 0.25 mmol/l of calcium, glucose did not influence somatostatin release while the presence of the sugar diminished basal and glibenclamide-induced glucagon secretion. The present data confirm the requirement of extracellular calcium for A, B and D cell secretion, demonstrating different calcium dependencies for the cell types and indicate that this dependency can, in part, be modulated by glucose.     

Parathyroid hormone-induced calcium efflux from isolated renal cortical tubules: evidence for cyclic AMP mediation.

Effects of parathyroid hormone (PTH) upon cyclic AMP and calcium efflux in isolated renal cortical tubules from hamsters were investigated. PTH caused a rapid rise in cyclic AMP levels, temporally preceding an increase in calcium efflux. Increases in both cyclic AMP levels and calcium efflux were noted over an identical PTH concentration range 0.007--0.7 U/ml). Other peptide hormones tested which had no effect upon cyclic AMP levels did not enhance efflux of calcium. The phosphodiesterase inhibitor methyl isobutylxanthine (MIX) was utilized in other studies to potentiate the cyclic AMP response, and produce a range of cyclic AMP concentrations in response to PTH. In these experiments a range of calcium efflux responses was noted which closely paralleled changes in cyclic AMP. Direct addition of cyclic AMP or dibutyryl cyclic AMP to isolated renal tubules caused increased efflux of calcium, while addition of 5'-AMP did not. These results indicate a role for cyclic AMP as a mediator of PTH-induced calcium efflux in this system and suggest that cyclic AMP may mediate the action of this hormone in enhancing renal conservation of calcium in vivo.     

Chronic and acute effects of forskolin on isolated thyroid cell metabolism

The chronic treatment (2 days or more) of cultured thyroid cells with 1-10 microM forskolin (forskolin-treated cells) sensitizes the response of adenylate cyclase to further acute stimulation by 100 microM forskolin or 10 mU/ml thyrotropin (TSH). This positive regulation, similar to that produced by 0.1 mU/ml TSH (TSH-treated cells), is obtained between 2 and 3 days of culture. The acute response to TSH or forskolin of cells treated for 4 days with forskolin increases with the concentration of forskolin present during the chronic treatment. This result is different from that obtained after a chronic treatment with TSH which induces refractoriness beyond 0.1 mU/ml. These cells are then desensitized to TSH but not to forskolin. When both agonists are mixed together, their acute effect is additive on control, TSH- and forskolin-treated cells. The chronic treatment of cultured thyroid cells with 1-10 microM forskolin produces, just like 0.1 mU/ml TSH, a chronic phospholipid effect characterized by enhanced incorporation of 32Pi into phosphatidylinositol (PI) and phosphatidic acid. The acute challenge of these cells with 100 microM forskolin evokes a reverse phospholipid effect, i.e. a decreased incorporation of 32Pi into PI. The acute stimulation of TSH-treated cells with TSH produces a reverse phospholipid effect whereas the acute stimulation of forskolin-treated cells with TSH gives a normal phospholipid effect as it does on control cells. These results show that the observed effects of TSH on cAMP accumulation and phospholipid turnover are not independent and are regulated in an inverse reciprocal pattern.     

The role of calcium in the induction of refractoriness to cyclic AMP stimulation by TSH

An initial exposure of beef thyroid slices to 25 mU/mL thyroid-stimulating hormone (TSH) for two hours induces a diminished stimulation of cyclic adenosine monophosphate (AMP) production upon subsequent readdition of TSH but does not modify the effect of prostaglandin E1 (PGE1). Incubation of thyroid slices in calcium-free buffer with or without 2 mmol/L ethylene glycol bis (beta-aminoethyl ether)--N,N' = tetracetic acid (EGTA) prevented desensitization induced by TSH and PGE1, to the subsequent stimulation by TSH and PGE1, respectively, despite the presence of calcium in subsequent incubations. TSH-induced desensitization was not modified by increasing the calcium concentration up to 50 mmol/L in the initial incubation. However, the stimulatory effect of TSH upon cyclic AMP levels was decreased as the calcium concentration in the first incubation was increased. In the presence of at least 1 mmol/L calcium, an initial incubation of thyroid slices with 20 mumol/L ionophore A-23187 decreased the stimulation of cyclic AMP by 25 mU/mL TSH added to the slices for the first time during a subsequent incubation. Under these conditions, A-23187 had no effect on PGE1 stimulation of cyclic AMP. These results indicate that calcium may play a role in the TSH-induced, but not PGE1, desensitization of cyclic AMP formation.     

Cellular cyclic nucleotides and enzyme secretion in the pancreatic acinar cell.

Cellular levels of cAMP and cGMP were measured in guinea pig pancreatic lobules incubated in vitro, during basal or stimulated secretion. Stimulation with optimal concentrations of carbamylcholine (carbachol) (10(-5) M), pancreozymin (0.1 unit/ml), and caerulein (10(-9) M) resulted within seconds in a sharp rise in cGMP levels, from five to more than 20 times that of basal levels. cAMP levels did not change significantly. cGMP increases were maximal at 2 min then subsided by 4-7 min to a plateau about two to three times that of basal level. This plateau was maintained for the duration of the secretagogue stimulus. Removal of the carbachol stimulus resulted in a rapid decrease in cGMP levels to that of the basal state. The cellular cGMP levels observed within the first 2 min of stimulation correlated closely with the dose of carbachol and the secretory response. Atropine at 10(-4) M blocked the cGMP elevation due to carbachol but not that due to pancreozymin, while carbonyl cyanide m-chlorophenyl hydrazone, an uncoupler of oxidative phosphorylation, blocked the response to both secretagogues. Similar though less extensive findings were observed using rabbit pancreatic lobules incubated in vitro. High concentrations (10(-2)-10(-3) M) of the dibutyryl and 8-bromo analogues of both nucleotides were effective, though suboptimal, secretagogues. In the case of the cAMP analogues, the secretory response was associated with a rise in endogenous cGMP levels, similar to that observed during suboptimal carbachol stimulation. These findings suggest that cGMP may be an intracellular mediator in the process of stimulus secretion coupling in the acinar cell of the exocrine pancreas.