Forskolin stimulation of adenylate cyclase in human thyroid membranes

Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with half-maximal stimulation occurring at 5-10 microM. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p- (above 1 microM). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.     

Time-related thyroid stimulation by thyrotropin and thyroid-stimulating antibodies, as measured by the cytochemical section bioassay

The time course of response of thyroid sections in the cytochemical bioassay to either TSH or thyroid-stimulating antibodies is bell shaped. The maximal staining for lysosomal naphthylamidase activity achieved was found to be the same regardless of the dose of stimulator applied; however, the rate at which the maximum was attained was dose dependent. Sections exposed to 10(-1) mU/liter TSH showed a maximal response at 120 sec, and those exposed to 10(-3) mU/liter TSH showed a maximal response at 210 sec. A similar dose-time effect was seen with immunoglobulin G from a thyrotoxic patient. Thus, by selecting a specific exposure time, a dose-response curve to the stimulator was obtained. A dose-response curve to a range of concentrations from 10(-4)-10(-1) mU/liter TSH was obtained by exposing sections to the hormone for 90 sec. TSH (10(-3) mU/liter) produced a response significantly different (P less than 0.0025) from the control. However, 10(-5) mU/liter TSH produced a response significantly different (P less than 0.0025) from the control after an exposure time of 180 sec, and the range of the dose-response curve at this exposure time was 10(-6)-10(-3) mU/liter TSH. Each point on these two dose-response curves was determined in quintuplicate, and precision profiles were constructed. The assay performed at 90 sec had a lower relative precision of 30% at a dose of 10(-1) mU/liter TSH, and at 180 sec, the best lower relative precision achieved was 80%. Thus, the sensitivity of the assay was improved by increasing the exposure time of the sections to TSH, but with a resultant loss of relative precision.     

Characterization of monoclonal antibodies raised against solubilized thyrotropin receptors in a cytochemical bioassay for thyroid stimulators

Selected clones of monoclonal antibodies from mice immunized with solubilized preparations of bovine TSH receptors have been characterized in a cytochemical bioassay (CBA) for thyroid stimulators. This assay is based upon quantification of changes in naphthylamidase activity of sections of guinea pig thyroids with use of a chromogenic substrate. Monoclonal 22A6 is a thyroid-stimulating antibody directed at a site within the TSH receptor. Thus, although it is a weak inhibitor of 125I-TSH binding to thyroid membranes, 22A6 is inhibited from binding to membranes by TSH, exhibits a more than additive agonist effect on adenylate cyclase activity when tested at low TSH concentrations in thyroid cells, and is a competitive antagonist of TSH enhancement of adenylate cyclase activity at high TSH concentrations. In the CBA, 22A6 is a stimulator whose maximal activity is obtained with 77 pg/ml (3-min exposure). Dose-response curves of a long acting thyroid stimulator (LATS)-B standard and 22A6 have slopes which are not significantly different; as anticipated, the response to LATS-B is inhibited by antihuman immunoglobulin G (IgG) and that due to 22A6 by antimouse IgG. In contrast to 22A6, monoclonal 11E8 is a relatively potent inhibitor of 125I-TSH binding as well as TSH stimulation of adenylate cyclase activity, while failing to act as a stimulator itself. 11E8 is itself inactive as a stimulator in the CBA over a wide dose range; it does, however, inhibit TSH stimulation in the CBA. This inhibition is abolished by antimouse IgG. The transient peak of response observed in time courses to TSH occurs later in the presence of 11E8. Unlike its effect on TSH 11E8 shows relatively low potency (greater than 10,000-fold lower) when inhibiting stimulation by the thyroid stimulating antibodies, 22A6 or LATS-B. Since this difference cannot be explained by quantitative differences in the ability of 22A6 or 11E8 to bind to thyroid membranes, the CBA data suggest that the stimulating antibodies, 22A6 and LATS-B, may interact with different determinants on TSH receptors then either TSH or the blocking antibody, 11E8. This also implies that in Graves' disease blocking antibodies may be incompletely expressed in the presence of stimulating antibodies, although they may be potent inhibitors of TSH binding, as measured in receptor assays.     

The application of a cytochemical bioassay to measure thyroid stimulating antibodies; a study of patients with euthyroid Graves' ophthalmopathy

Using a cytochemical bioassay for thyroid stimulators, we have studied in detail the responses to IgG preparations from sera of 11 patients with euthyroid Graves' ophthalmopathy. This assay is based upon changes in naphthylamidase activity in response to the addition of thyroid stimulators. A wide range of dilutions of each IgG preparation was tested, and bell-shaped dose response curves were obtained where thyroid stimulating antibody (TSAb) was present. Such dose-response curves, giving a maximum response at one dilution (higher or lower concentrations giving a decreased or nil response) are characteristic of this assay. Results were expressed as a titer which is defined as the reciprocal of the dilution giving the maximum response for a given sample. Our routine strategy for titer determination for a given IgG preparation, including the use of "negative" and "positive" controls is described. Retesting of IgG's in separate bioassays showed that the titers were reproducible. Serum from one of the 11 patients was TSAb negative, i.e. did not provoke a response at any dilution tested in this ultrasensitive bioassay. The others were TSAb positive with a wide range of titers (1:10(2) to 1:10(8)). In addition one patient (initial titer 1:10(5)) was plasmapheresed, first in the absence of immunosuppression, when rebound resulted in a titer of 1:5 X 10(7), and secondly in combination with immunosuppression when the titer was reduced to less than 1:10(2). These large changes in titer for samples from one patient were consistent with the strikingly wide ranges of titers found for the 10 other patients.     

VIP stimulation of beta-naphthylamidase activity in guinea-pig thyroid sections

Subsequent to the discovery of vasoactive intestinal peptide (VIP) in the thyroid gland, VIP has been shown to stimulate various thyroid functions. The site of interaction of VIP with the thyroid follicular cell is at present not known, and this study has used the ultrasensitive cytochemical bioassay (CBA) for thyroid stimulators to investigate this further. Exposure of thyroid sections for 3 min to VIP resulted in increased naphthylamidase activity, with half-maximal response observed at 3 X 10(-13) M VIP. This response to such low doses of VIP is consistent with the CBA being ultrasensitive to other thyroid stimulators e.g. TSH, thyroid stimulating antibodies and forskolin. The response to VIP was abolished by rabbit anti-VIP antiserum. The dose-response curve to VIP was bell-shaped (as with the other stimulators), maximal stimulation occurring at 10(-12) M VIP. In contrast, however, to other thyroid stimulators, namely TSH, LATS-B and 3 monoclonal stimulating antibodies, whose ascending limbs of the dose-response curves extended over 3-4 orders of magnitude, the VIP curve rose rapidly from basal to maximal tissue stimulation from 10(-13) to 10(-12) M VIP, i.e. one order of magnitude. This unusual dose-response curve to VIP was parallel to that produced by forskolin. 11E8, a monoclonal 'blocking' antibody which is a potent inhibitor of TSH stimulation, did not 'block' forskolin stimulation, consistent with the belief that forskolin acts at a post-receptor site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of monoclonal antibodies derived from lymphocytes from Graves' disease patients in a cytochemical bioassay for thyroid stimulators

Monoclonal antibodies 208F7 and 307H6, derived from Graves' lymphocytes, were previously shown to stimulate thyroid function. We characterized these antibodies in the ultrasensitive cytochemical bioassay for thyroid stimulators. Bell-shaped dose-response curves were obtained for both antibodies, confirming their actions as thyroid stimulators; 307H6 was 10(7) times more potent than 208F7, and the ascending limb of the response curve to 307H6 was not significantly different from that of a reference preparation of thyroid-stimulating antibodies, namely LATS-B. Stimulation by both 208F7 and 307H6 was inhibited by antihuman, but not antimouse, immunoglobulin. Stimulation by 208F7, but not 307H6, was inhibited by 11E8 (a monoclonal antibody raised against the TSH receptor), which is a relatively potent inhibitor of TSH, but not thyroid-stimulating antibodies. These findings together with previous observations on the interactions of 208F7 and 307H6 with thyroid cells in both the presence and absence of TSH and of 208F7 and 307H6 with solubilized thyroid membrane components are summarized in a model relating the appropriate epitopic regions of the TSH receptor.     

Further studies on the response of a cytochemical bioassay to thyroid stimulators, using reference preparations of thyrotropin and long acting thyroid stimulator

The fundamental response of this assay is shown to be a shortening of a latency period prior to a peak of staining after tissue sections have been exposed to either TSH or LATS. The magnitude of the peak for a given tissue is both stimulator and dose independent. Simultaneous exposure to a combined dose of the two stimulator resulted in a further shortening of the latency period, and no evidence of two peaks was observed in the presence of both stimulators. Dose-response curves after exposure to reference preparations of the two stimulators for the same time period (180 s.) were parallel: this indicates that given incremental doses of either stimulator result in indistinguishable increases in the fundamental response of this assay.     

Forskolin: effects on cyclic AMP and contractile function in the isolated rat and guinea pig heart

The new inotropic agent, forskolin, is thought to act by stimulation of adenyl cyclase at a site remote from the beta-receptor. In this study we have characterized the action of forskolin in isolated, perfused rat and guinea-pig hearts. In both species, forskolin increased heart rate, left ventricular pressure and tissue cyclic AMP in a concentration-dependent manner. Significant responses were obtained with a minimum concentration of 2 X 10(-9)M forskolin in the rat and 2 X 10(-8)M in the guinea-pig. In both species maximal effects were observed with 2 X 10(-6)M forskolin. For any given forskolin concentration, the increases in cyclic AMP were greater in the rat heart than in the guinea-pig heart. In the rat heart, two beta-adrenoceptor blocking agents (1.6 X 10(-6)M propranolol or 2.6 X 10(-6)M timolol) were both able to reduce the increases in function and tissue cyclic AMP content caused by forskolin (2 X 10(-7) and 2 X 10(-8)M). However, no inhibition by beta-blockade was observed in hearts from catecholamine-depleted (reserpinized) animals or in hearts treated with high concentrations (2 X 10(-6)M) of forskolin. These results indicate that catecholamines may in some way be able to potentiate the actions of forskolin.     

Electrical responses of guinea pig coronary artery to transmural stimulation

Isolated segments of the guinea pig circumflex coronary artery were stimulated transmurally with brief duration (0.05 msec) pulses while recording intracellularly. Resting membrane potential was -60.3 +/- 4.3 mV (n = 66). Transient depolarizations (TDs) graded in amplitude with stimulus intensity were elicited with single stimuli. An action potential of 47 +/- 5 mV was superimposed upon the TD above a threshold membrane potential of -48 +/- 4 mV. Following the spike, a period of slow repolarization requiring 77 +/- 33 seconds was observed. Repetitive stimulation led to summation of TDs and a slow depolarization (less than or equal to 21 mV) that persisted for up to 90 seconds. The TD was reduced in amplitude when the membrane was depolarized with increased [K]o and enhanced when the membrane was hyperpolarized by adding potassium back to a potassium-depleted tissue. Phentolamine (10(-5) M), prazosin (10(-6) M), guanethidine (2 X 10(-5) M), alpha,beta-methylene ATP (2 X 10(-5) M), tetrodotoxin (10(-6) M), and 10-20 mM norepinephrine did not block TDs, action potentials, or slow depolarization. TDs were abolished following cold storage (4 degrees C) for 4 days and significantly reduced in a 2.25-mM CdCl2 solution with 0.25 mM CaCl2. Acetylcholine (10(-8) M to 10(-5) M) produced membrane hyperpolarization and reduced the amplitude of TDs and altered their time course. Atropine (10(-5) M) blocked the effects of acetylcholine but had no effect on the response to nerve stimulation. These observations indicate that TDs exhibit some, but not all, of the properties characteristic of excitatory junction potentials obtained with release of neurotransmitter substances in other vessels. If these TDs are neural events, they must involve a transmitter other than norepinephrine, acetylcholine, or ATP.     

Forskolin and thyrotrophin stimulation of rat FRTL-5 thyroid cell growth: the role of cyclic AMP

The adenylate cyclase stimulator forskolin increases intracellular cyclic AMP (cAMP) in rat FRTL-5 cells within minutes and, after a lag phase of 20-24 h, an increase of cells in metaphase is seen. The dose-response relationships were similar in both systems, with significant increases in the number of metaphases observed at approximately 0.1 mumol/l and a doubling of cAMP levels at 1 mumol/l, whilst doses of 0.1 mmol/l and above proved cytotoxic. An involvement of intracellular cAMP as a positive intermediate in cell division was further suggested by the finding that a low dose of forskolin (0.1 mumol/l) potentiated TSH stimulation of mitosis. Isobutyl methyl xanthine (IBMX), a phosphodiesterase inhibitor, also acted as a mitogen and potentiated TSH action. Moreover, the simultaneous inclusion of low doses of IBMX and forskolin additionally potentiated TSH stimulation of mitosis. An analogue of cAMP, dibutyryl cAMP, also stimulated mitosis and acted over a restricted dose range, with maximal stimulation at 1 mmol/l. We conclude that cAMP may act as a positive signal for FRTL-5 thyroid cell proliferation.     

Relaxin detected by immunocytochemistry and northern analysis in the mammary gland of the guinea pig

Relaxin is a product of the endometrial gland cells in the guinea pig. Mammary tissue was collected from intact cyclic animals, on days 35 and 63 of pregnancy, days 5 and 21 of lactation, and day 28 postpartum. Tissue was collected from six cyclic hysterectomized animals. Sections of mammary gland were immunostained with the avidin-biotin immunoperoxidase method, and antisera to porcine relaxin which were raised to different preparations in different laboratories. Light uniform staining was evident in the cytoplasm of all of the cuboidal epithelial cells forming the mammary duct system in cyclic animals; mammary gland from hysterectomized animals showed similar staining. At midpregnancy light staining was seen in some epithelial cells, and by late pregnancy there was only faint staining. On day 5 of lactation there was intense and uniform staining throughout the epithelial cells of the alveoli. By day 21 of lactation the cells still immunostained for relaxin, but on day 28 postpartum there was a return to the cyclic staining pattern. Endometrium from animals at 55-60 days of pregnancy and mammary gland from day 6 of lactation were used for poly(A)+ RNA isolation and Northern analysis. Three 48-mer oligonucleotide probes were used. Poly(A)+ RNA from endometrium of late pregnant guinea pigs and from the mammary gland in lactation hybridized with the same two probes and failed to hybridize with a third under moderate stringency conditions. The results suggest that the major source of relaxin in the guinea pig is sequential, the pregnant uterus and the lactating mammary gland. The local and/or systemic significance is not known.     

Activation of chloride current by purinergic stimulation in guinea pig heart cells.

Single atrial cells from guinea pig heart were voltage-clamped using the whole-cell configuration of the patch-clamp technique under conditions in which most of the ionic and exchange currents known in cardiac cells were minimized. Extracellular 5 or 50 microM ATP activated a Cl- current, in addition to a rapidly desensitizing cation-selective current. A nonhydrolyzable ATP analogue, adenosine-5'-O-(3-thiotriphosphate) (50 microM), also evoked these two currents, indicating involvement of purinoceptors rather than ecto-ATPase on the membrane. ADP, AMP, and adenosine were also effective in inducing the Cl- current, showing no clear order of potency for the purinoceptor subtypes involved. The purinoceptor-activated Cl- current, like the beta-catecholamine-cAMP-dependent cardiac Cl- current, showed outward rectification and time independence.     

Clinical experience with a human thyroid cell bioassay for thyroid-stimulating immunoglobulin

A sensitive, specific, and practical bioassay for thyroid-stimulating immunoglobulin (TSI) is now available for clinical use. Fifty-seven of 61 patients with untreated hyperthyroid Graves' disease were TSI positive (sensitivity, 93%). TSI was undetectable in all normal subjects and in patients with Hashimoto's thyroiditis (without concurrent Graves' ophthalmopathy), nontoxic goiter, and toxic nodular goiter (specificity, 100%). The prevalence of TSI in serum declined after therapy, particularly during methimazole or propylthiouracil treatment. TSI correlated well with relapse or remission after antithyroid drug therapy. All 12 patients who were TSI negative at the time of discontinuing antithyroid drug therapy remained in remission (average follow-up of 10 months). TSI values in Graves' disease correlated better with thyroid dysfunction than with ophthalmopathy. Prenatal TSI activity tended to be higher in mothers of infants who developed neonatal Graves' disease than in at-risk mothers who delivered normal infants. However, there was considerable overlap between the two groups.     

Release of individual enzymes from guinea pig pancreas following stimulation with CCK-8 or CCK-33

This paper compares the effects of prolonged stimulation with CCK-33 or CCK-8 on the secretion of individual enzymes from the pancreas of the anaesthetised guinea-pig. Quantitative analysis of the individual secretory proteins was achieved using a newly developed, reversed-phase, high-performance, liquid chromatography technique. Using this system, it was possible to separate and quantify each of the nine major proteins present in a small sample of pancreatic juice in 40 min. Because spontaneous pancreatic secretion was very slow in the anaesthetised guinea-pig, the cholecystokinin (CCK) peptides were administered against a background infusion of secretin. Secretin alone produced a small rise in total protein release with the secretion of all individual proteins being increased in parallel. Doses of CCK-33 and CCK-8 which increased total protein secretion by a similar amount had contrasting effects on the release of individual proteins. CCK-33 produced a parallel release of all proteins over a period of 2 h. By contrast, after 1-h stimulation with CCK-8, the secretion of proteins became nonparallel. These data suggest that CCK-33 and CCK-8 may provoke slightly different intracellular responses within the pancreatic acinar cell.