The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration.

The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4--17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4--9 days depolarized from --29.6 +/- 1.7 (mean +/- S.E.) to --19.3 +/- 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in the electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.     

Iodide induced suppression of thyrotropin-stimulated adenosine 3',5'-monophosphate production in cat thyroid slices.

Cat thyroid slices were studied to investigate their responsiveness to thyrotropin stimulation of cyclic AMP accumulation. Ovine and bovine thyrotropin, in the presence of 2.5 mM aminophylline, induced a dose-dependent increase in the cyclic AMP content of cat thyroid tissue. Half-maximal stimulation of cyclic AMP accumulation was obtained at a thyrotropin concentration of 1-2 mU/ml. The maximal effect of thyrotropin was observed at 10 mU/ml, and was associated with a mean 77 +/- 19-fold increase in thyroidal cyclic AMP. Preincubation of cat thyroid tissue for 2 h with 50 micron NaI resulted in an impairment in the subsequent ability of thyrotropin to enhance cyclic AMP accumulation, without altering the level of cyclic AMP in tissues not exposed to the hormone. Preincubation alone was without effect on thyrotropin stimulation of cyclic AMP, and the inhibitory effect of iodide was prevented by addition of 3 mM methimazole to the preincubation medium. In addition, the time course of thytrotropin stimulation of cyclic AMP accumulation in cat thyroid slices was not significantly altered by the preincubation with excess iodide. These studies provide additional evidence that excess iodide inhibits the adenylate cyclase-cyclic AMP system in thyroid tissue.     

The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration

The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4–17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4–9 days depolarized from ?29.6 ± 1.7 (mean ± S.E.) to ?19.3 ± 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 1. Negative regulation.

Isolated porcine thyroid cells, cultured in the presence of thyrotropin (greater than or equal to 0.25 mU/ml) or prostaglandin E2 (greater than or equal to 0.1 micron), showed decreased adenosine 3':5'-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin E2 stimulation, respectively. Kinetics of the refractory process to thyrotropin and prostaglandin E2 are different: (a) maximal refractoriness to prostaglandin E2 was attained after 2--6 h exposure to prostaglandin E2 while refractoriness to thyrotropin was maximal only after 12--24 h; (b) the degree of refractoriness to prostaglandin E2 was much greater than that to thyrotropin. Refractoriness to thyrotropin or prostaglandin E2 is characterized: by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 micron prostaglandin E2), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility. This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3':5'-monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin E2 by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Electrophysiological studies on the cultured cells obtained from transplantable pancreatic carcinoma in Syrian golden hamsters

A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5 +/- 1.8 mV (S.E.) (n = 13), while the mean resting input resistance was 21.2 +/- 4.3 M omega (S.E.) (N = 13). Dibutyryl cyclic AMP (2 X 10(-3)M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.     

Electrophysiological responses of crayfish oocytes to biogenic amines

Intracellular recordings were made from immature, growing oocytes of the crayfish Pacifastacus leniusciulus. Oocytes had a relatively negative resting potential of -74.7+/-2.2 mV (n=26; range -53 to -90) and a mean input resistance of 0.86+/-0.19 MOmega (n=22; range 0.17-3.3). Octopamine induced a long-lasting response involving biphasic changes in input resistance, together with bi- or multiphasic changes in membrane potential. The resistance-decreasing phase involved (in different oocytes) membrane hyperpolarization, depolarization or both. The resistance-increasing phase was usually a depolarization. The hyperpolarizing form of the resistance-decreasing response, and the depolarizing resistance-increasing response reversed in polarity at membrane potentials of (respectively) -90 and -92 mV, suggesting increases and decreases in K(+) conductance underly the biphasic changes in input resistance. The threshold concentration for the response was remarkably low (>10(-12) M) and showed little or no dose-dependence over the concentration range 10(-12)-10(-6) M. Similar responses were evoked by dopamine and serotonin (at 10(-9) M), although a higher proportion of oocytes responded to octopamine and/or dopamine than to serotonin.     

Cyclic AMP in the thyroid of the rat fed prophylthiouracil: in vitro unresponsiveness to thyrotropin.

Fragments of thyroid from rats fed Purina +0.1% propylthiouracil were incubated in vitro and the concentration of cyclic AMP measured. The normal gland showed a 3-fold increase in cyclic AMP with 50 mU thyrotropin per ml or 10(-4) M prostaglandin E1; tissue from rats fed propylthiouracil for 10 days to 6 months responded to prostaglandin E1 but not to thyrotropin. Feeding Purina without propylthiouracil for 1 month after 5 months of goitrogen restored thyrotropin-responsiveness, as did, to a lesser extent, injection of triiodothyronine, 50 mug twice daily for 5 days.     

Adenosine 3':5'-monophosphate metabolism and turnover in dog thyroid slices.

The metabolism and turnover of adenosine 3':5'-monophosphate (cyclic AMP) have been studied in intact thyroid cells incubated in vitro. Thyroid slices have been stimulated by 1 mU thyrotropin/ml, then washed with buffer, or with buffer containing thyrotropin antibody, or trypsin so as to cut off the stimulation. The decline of cyclic AMP levels has been followed and the time required to decrease this level to half of the initial value estimated. Computer simulation taking into account the penetration of trypsin in the slices, the kinetics of thyrotropin inactivation and the relation between thyrotropin concentration and cyclic AMP concentration at the steady state has made it possible to estimate the true cellular half-life of cyclic AMP in the stimulated cell to 1 min 50 s. The method provides an experimental approach to the demonstration in intact cells of effective on cyclic AMP disappearance. The methodology of the calculation of half-life and turnover from such data is discussed.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into follicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 μU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 μU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles

The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation.     

Screen-printed amperometric microcell for proline iminopeptidase enzyme activity assay

A microfabricated amperometric microcell was designed and used for the determination of proline iminopeptidase (PIP) enzyme activity in 2-10-microl samples. The measurements were made in the range of 10.3-841.5 mU/ml enzyme activities. The sensitivity of the determinations was between - 0.0195 and - 0.0203 microA ml/mU per min. The coefficient of variation of the determined values ranged between 2.8 (at 561.2 mU/ml) and 24.1% (at 10.3 mU/ml). The microcell was manufactured on an alumina substrate using screen-printed graphite working and Ag/AgCl reference electrodes. Elevated PIP activity in the vaginal fluid is a biochemical indicator of bacterial vaginosis. The method is appropriate to differentiate between normal (66+/-145 mU/ml) and elevated, diseased (704+/-145 mU/ml), values.     

Protein-bound cAMP, total cAMP, and protein kinase activation in isolated bovine thyrocytes.

Thyrocytes isolated from bovine thyroid tissue were subjected to thyrotropin treatment and analyzed for total cAMP, protein bound cAMP and protein kinase activation. The concentration of thyrotropin (～4 mU/ml) required for half-maximal elevation of total cAMP was 20 times greater than the concentration required for half-maximal elevation of protein kinase activity and bound cAMP levels (～0.2 mU thyrotropin/ml). In thyrocytes, bound cAMP and protein kinase activation showed a linear, one to one relationship indicating that bound cAMP obtained with the charcoal procedure is largely or completely identical with R·cAMP.     

Blood erythropoietin level in female reproductive age (can the reproductive tissue be the source of erythropoietin in blood?)

Blood erythropoietin level was investigated in females of reproductive age, who had normal peripheral red blood findings. It was determined that blood erythropoietin level was 1.5-2 times higher (20.04 +/- 2.10; 28.57 +/- 5.87 mU/ml) in examined females with increased mitotic activity and hyperplasia signs of cervical squamous epithelia compared with female having normal cervix (12.73 +/- 0.96 mU/ml). In females with chronic cervicitis, blood erythropoietin level was decreased (8.87 +/- 1.72 mU/ml).     

Muscle glucose uptake of obese Zucker rats trained at two different intensities

Exercise training reduces the muscle insulin resistance of the obese Zucker rat. The purpose of the present study was to determine whether the magnitude of this training response is exercise intensity specific. Obese Zucker rats were randomly divided into sedentary (SED), low-intensity (LI), and high-intensity (HI) exercise groups. For the LI rats, exercise training consisted of running on a rodent treadmill at 18 m/min up an 8% grade for 90 min. Rats in the HI group ran at 24 m/min up an 8% grade for four 17-min bouts with 3 min between bouts. Both exercise groups performed the same amount of work and trained 5 days/wk for 7 wk. To evaluate muscle insulin resistance, rat hindlimbs were perfused for 30 min with perfusate containing 6 mM glucose (0.15 mu Ci of D-[14C(U)] glucose/ml) and either a maximal (10.0 mU/ml) or a submaximal (0.50 mU/ml) insulin concentration. Perfusions were performed 48-56 h after the last exercise bout and a 12-h fast. In the presence of 0.5 mU/ml insulin, the rate of muscle glucose uptake was found to be significantly faster for the HI (9.56 +/- 0.66 mumol.h-1.g-1) than for the LI (7.72 +/- 0.65 mumol.h-1.g-1) and SED (6.64 +/- 0.44 mumol.h-1.g-1) rats. The difference in glucose uptake between the LI and SED rats was not significant. In the presence of 10.0 mU/ml insulin, the rate of glucose uptake was significantly faster for the HI (16.43 +/- 1.02 mumol.h-1.g-1) than for the LI rats (13.76 +/- 0.84 mumol.h-1.g-1) and significantly faster for the LI than for the SED rats (11.02 +/- 0.35 mumol.h-1.g-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic mechanisms of two types of on-center bipolar cells in the carp retina. I. The responses to central illumination.

Properties of the depolarizing response of on-center bipolar cells to a light spot stimulus were studied in the carp retina. On-center bipolar cells were classified into two types, cone-dominant and rod-dominant, according to their major input from cones and rods. Cone-dominant bipolar cells responded to spectral light with the maximum amplitude near 625 nm, suggesting major input from red cones. The response was accompanied by a resistance increase and showed a reversal potential at -63 +/- 21 mV when the membrane was hyperpolarized by current. The results suggest that the photoresponse of cone-dominant cells is due to a decrease of gK and/or gCl, membrane conductances to potassium and chloride, respectively. Rod-dominant bipolar cells responded to spectral light with the maximum amplitude near 525 nm under scotopic conditions and near 625 nm under photopic conditions, providing evidence that they receive input from rods and red cones. In the scoptopic condition their response was accompanied by a resistance decrease and showed a reversal potential at 29 +/- 13 mV, whereas in the photopic condition the response in most of them was accompanied by a resistance increase, at least in their part and showed a reversal at -53 +/- 11 mV. The results suggest that the photoresponse activated by rod input is due to an increase in gNa. In the mesopic condition rod-dominant cells showed complex electrical membrane properties as the result of electric interaction between the above two differnt ionic mechanisms activated by rod and cone inputs.     

Decrease of erythropoietin level by human recombinant tumour necrosis factor alpha (hrec TNFalpha) in patients with advanced cancer

Anaemia is a frequent complication of chronic inflammation, infectious diseases and cancer. Inappropriate erythropoietin production is regarded as one of the main causative factors responsible for the occurrence of anaemia. The pathogenesis of TNFalpha induced-anaemia has not been fully clarified yet and its influence on hematopoiesis has been suggested. We performed a clinical study to access the influence of hrec TNFalpha administration on plasma EPO concentration and the degree of anaemia in patients with advanced solid tumours for whom no other kind of therapy but palliative treatment was available. All these patients exposed mild anaemia (HT 36.1 +/- 1.0%). Plasma EPO was estimated at 8 a.m. before and after 5 days of TNFalpha therapy with a dose of 75 pg/day iv (cycle I). Two weeks later plasma EPO was estimated again before and after 5 days of TNFalpha administration of a double dose (150 microg/day) (cycle II). The control group comprised 8 non-cancer patients (5M/3F, age 48.5 +/- 6yr) with the same degree of anaemia (HT 36 +/- 1.1%) due to haemorrhage. In the control group the plasma EPO level was significantly higher (54.2 +/- 8 mU/ml) than in cancer patients before cycle I (17.1 +/- 2.5 mU/ml) and II (14.6 +/- 3.8 mU/ml) respectively.TNF administration was followed by a significant decline of plasma EPO both after the first (17.1 +/- 2.5 vs 9.0 +/- 1.5 mU/ml) and second cycle (14.6 +/- 3.8 vs 8.4 +/- 2.0 mU/ml) of TNF treatment. CONCLUSIONS: Patients with solid cancer and mild anaemia are characterised by inappropriate low plasma EPO concentration. Therapy with TNFalpha exerts a suppressive effect on EPO secretion in these patients.     

Impaired insulin action in primary hyperaldosteronism

The presence of insulin resistance is frequently found in essential hypertension. There are, however, only sparse data with respect to the potential presence of insulin resistance in patients with secondary hypertension. We have therefore undertaken a study to reveal the potential occurrence of insulin resistance in primary hyperaldosteronism (PH). The hyperinsulinemic euglycemic clamp technique together with the evaluation of insulin receptor characteristics were used to study insulin resistance in 12 patients with PH. The measured parameters were compared to normal values in control subjects. We have found a significantly lower glucose disposal rate (M, micromol/kg/min) (18.7+/-6 vs. 29.3+/-4), decreased tissue insulin sensitivity index (M/I, micromol/kg/min per mU/l x100) (23.7+/-9.8 vs. 37.5+/-11.6) and also lower metabolic clearance rate of glucose (MCRg, ml/kg/min) (3.8+/-1.5 vs. 7.0+/-1.1) in patients with primary hyperaldosteronism. The insulin receptor characteristics on erythrocytes did not differ in primary hyperaldosteronism as compared to control healthy subjects. We thus conclude that insulin resistance is also present in secondary forms of hypertension (primary hyperaldosteronism) which indicates the heterogeneity of impaired insulin action in patients with arterial hypertension.     

Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.