Adenylate cyclase-cAMP System Research Articles

An Endosomal Acid-Regulatory Feedback System Rewires Cytosolic cAMP Metabolism and Drives Tumor Progression.

Endosomal pH regulator NHE9 actively controls cytosolic Ca2+ levels to downregulate the adenylate cyclase-cAMP system, enabling colorectal cancer cells to acquire hybrid E/M characteristics and promoting metastatic progression.

Extra- and Intracellular Mechanisms of Changes in Erythrocyte Aggregation

Reversible aggregation is a form of erythrocyte behavior. The presence of α- and β-adrenergic receptors on erythrocyte membranes suggests that aggregation can be influenced by agonists of these receptors. Human erythrocyte aggregation (EA) was studied in the presence of α- and β-agonists of adrenergic receptors (in the concentration range from 10−6 to 10−8 M). The α2-agonist clonidine stimulated EA most strongly (by 163%, P < 0.01). The cell reaction decreased from the α-agonist clonidine to the β-agonist metaproterenol. The phosphodiesterase inhibitor papaverine and the penetrating cAMP analogue dibutyryl cAMP (dB-cAMP) increased the intracellular cAMP and decreased EA by 46–50% (P < 0.05). Clonidine-induced stimulation of EA sharply decreased upon erythrocyte incubation in the presence of clonidine + dB-cAMP and became even lower than in the control. Thus, α-agonists of adrenergic receptors markedly stimulated EA. The adenylate cyclase-cAMP system is likely involved as an intracellular signaling pathway.

Relief from glucose-induced over-stimulation sensitizes the adenylate cyclase-cAMP system of rat pancreatic islets.

Hyperglycemia impairs beta-cell function. This effect is partly exerted by beta-cell over-stimulation by mechanisms that are not completely clarified. We have presently investigated whether over-stimulation alters the responsiveness of the islet adenylate cyclase-cAMP system. Effects of over-stimulation were assessed from comparisons in rat pancreatic islets after stimulation by culture for 22 h with high (27 mM) glucose or after the additional presence of diazoxide which reversibly blocks secretion. Islet ATP levels were similar under both conditions. Forskolin increased islet cAMP levels dose-dependently after culture under both conditions; however, the cAMP responses to forskolin were enhanced by the previous co-presence of diazoxide: by 354, 183 and 168% respectively in the presence of 0.1, 1.0 and 25 microM forskolin (P<0.05) or less for the effect of diazoxide. Enhancement was not diminished ! by Ca(2+ )omission during final incubations, nor by blocking Gi proteins with pertussis toxin (0.1 microgram/ml). Enhancement was dependent on the glucose concentration during culture, i.e. co-culture with diazoxide at a non-stimulatory concentration of glucose (6.0 mM) failed to affect the subsequent cAMP response to forskolin. Acute administration of glucose (16.7 mM) failed to increase islet cAMP content after culture at high glucose only, whereas a modest (about 20%) but significant stimulation was seen after co-culture with diazoxide. Co-culture with diazoxide left-shifted the insulin dose-response to a cAMP analogue 5,6-dichloro-1-beta-d> -ribofuranosyl-benzimidazole-3',5'-cyclic monophosphorothioate. We conclude that over-stimulation importantly modifies the generation of cAMP, and also affects the insulin-releasing effect of the cyclic nucleotide.

Role of cAMP in activation of ischemically sensitive abdominal visceral afferents

A number of metabolites produced during abdominal ischemia can stimulate and/or sensitize visceral afferents. The precise mechanisms whereby these metabolites act are uncertain. Other studies have shown that the adenylate cyclase-cAMP system may be involved in the activation of sensory neurons. Therefore, we hypothesized that cAMP contributes to the activation of ischemically sensitive abdominal visceral afferents. Single-unit activity of abdominal visceral C fibers was recorded from the right thoracic sympathetic chain in anesthetized cats before and during 7 min of abdominal ischemia. Forty-six percent of ischemically sensitive C fibers responded to intra-arterial injection of 8-bromo-cAMP (0.35-1. 0 mg/kg), an analog of cAMP, with responses during ischemia increasing from 0.50 +/- 0.06 to 0.84 +/- 0.08 impulses/s (P < 0.05, n = 11 C fibers). Conversely, an inhibitor of adenylate cyclase, 2', 5'-dideoxyadenosine (DDA; 0.1 mg/kg iv), attenuated ischemia-induced increase in activity of afferents from 0.66 +/- 0.10 to 0.34 +/- 0. 09 impulses/s (P < 0.05; n = 8). Furthermore, whereas exogenous PGE(2) (3-4 microg/kg ia) augmented the ischemia-induced increase in activity of afferents (P < 0.05, n = 10), treatment with DDA (0.1 mg/kg iv) substantially reduced the increase in discharge activity of afferents during ischemia, which was augmented by PGE(2) (1.45 +/- 0.24 vs. 0.70 +/- 0.09 impulses/s, -DDA vs. +DDA; P < 0.05) in six fibers. A time control group (n = 4), however, demonstrated similar increases in the activity of afferents with repeated administration of PGE(2). These data suggest that cAMP contributes to the activation of abdominal visceral afferents during ischemia, particularly to the action of PGs on activation and/or sensitization of these endings.

Inhibition of prostaglandin synthesis reduces cyclic AMP levels and inhibits chondrogenesis in cultured chick limb mesenchyme.

The present study investigated effects of inhibiting the synthesis of prostaglandins (PGs) on cyclic AMP concentrations and chondrogenesis in cultured chick limb mesenchyme. Indomethacin produced concentration-dependent inhibition of both PGE(2) synthesis and chondrogenesis over a concentration range of 50--200 microM. Half maximal inhibition of PGE(2) was achieved with 50 microM concentrations of the drug which also produced visibly reduced amounts of cartilage matrix in cell cultures as evaluated by Alcian green staining on day 6 of culture. The inhibitory effects of indomethacin on chondrogenesis were largely reversed by addition of 1 mM dibutyryl cAMP, indicating that cells could still respond to cyclic AMP stimulation. Endogenous levels of cyclic AMP, which increased by 6 fold during the six days of culture in control cells, did not increase significantly from dissociated cells at the time of plating (day 0) in indomethacin- treated cultures. The results indicate that inhibition of the prechondrogenic rise in PGE(2) concentrations in limb mesenchyme prevents the increase in cyclic AMP levels which occur during this same period resulting in inhibition of chondrogenesis. The data provide further support for the hypothesis that PGE(2), through its effects on the adenylate cyclase-cAMP system, plays an important role in the differentiation of cartilage.

Increase of urinary extracellular-superoxide dismutase level correlated with cyclic adenosine monophosphate

Extracellular superoxide dismutase (EC-SOD) is a secretory protein that is the major SOD isozyme in extracellular fluids. Plasma EC-SOD levels are distributed in two discrete groups with the rare group having an enzyme with glycine instead of arginine-213, which causes a 10-fold higher serum level. Within the common phenotype group, the urinary EC-SOD level was significantly correlated with the urinary excretion of N-acetyl-β- D-glucosaminidase (NAG), but not with serum EC-SOD. EC-SOD appears not to be leaked from the plasma by glomerular filtration, but rather to be secreted from the renal tubule or its surrounding tissues. The urinary EC-SOD level was also significantly correlated with the urinary cyclic adenosine monophosphate (cAMP) level. cAMP analogues and adenylate cyclase modulators significantly stimulated the expression of EC-SOD but not other SOD isozymes in cultured fibroblast cell lines. Moreover, injection of parathyroid hormone, in Ellsworth-Howard tests, increased urinary EC-SOD accompanied with the elevations of urinary cAMP and NAG. Together these observations suggest that factor(s) that stimulate the adenylate cyclase-cAMP system regulate the urinary EC-SOD level.

Effects of various phosphodiesterase-inhibitors, forskolin, and sodium nitroprusside on porcine detrusor smooth muscle tonic responses to muscarinergic stimulation and cyclic nucleotide levels in vitro

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers involved in the regulation of contractility in various smooth muscle organs including detrusor smooth muscle. They are synthesized by activation of adenylate and guanylate cyclases, respectively, and inactivated by phosphodiesterases (PDEs). In order to delineate the intracellular regulation of porcine detrusor contractility by cyclic nucleotides and phosphodiesterases, functional organ bath studies and determinations of intracellular cyclic nucleotide contents were performed after incubation of porcine detrusor strips with forskolin (adenylate cyclase activator), sodium nitroprusside (guanylate cyclase activator), and various phosphodiesterase-inhibitors. Significant relaxant responses were achieved only by forskolin, the nonspecific phosphodiesterase-inhibitor papaverine, and the phosphodiesterase 1-inhibitor vinpocetine (62.4 +/- 5.6%, 73 +/- 4.3%, and 53 +/- 7.9%, respectively). Sodium nitroprusside and the selective PDE-inhibitors milrinone, rolipram, zaprinast, and dipyridamole were significantly less efficacious (26.9 +/- 3.9%, 15.5 +/- 3.8%, 15.3 +/- 3.0%, 13 +/- 4.0%, and 13.2 +/- 2.1%, respectively). Forskolin, papaverine, and vinpocetine elevated intracellular cAMP concentrations (7.3-, 1.9-, and 1.7-fold increase at 100 microM, respectively), whereas the other substances failed to enhance cAMP levels. cGMP levels were only increased by sodium nitroprusside (7.8-fold). The adenylate cyclase-cAMP system seems to be the more important signal transduction system involved in the relaxation of carbachol induced smooth muscle tone of the porcine detrusor. The role of the guanylate cyclase-cGMP system is less clear. In addition, the calcium/calmodulin-stimulated PDE I seems to be of major functional importance in regulating cAMP hydrolysis in the porcine detrusor smooth muscle in vitro.

Modulation of cyclic AMP production by strial marginal cells from gerbil in culture

To further investigate the role of marginal cells (MCs) in the secretion of endolymph and because of the limitations encountered in investigating these cells in vivo, we used primary cultures of MCs derived from explants of gerbil stria vascularis and investigated modulation of the adenylate cyclase-cyclic AMP system. After 10 days on type I collagen coated plastic dishes, a confluent monolayer of epithelial-like cells was obtained which exhibited the morphologic and immunohistochemical features of the native marginal cells. The cyclic AMP (cAMP) content was determined at 37°C, after 5 min of incubation with various agents, in the presence of a specific inhibitor of type III cAMP-dependent phosphodiesterase, RO 20–1724. The adenylate cyclase-cAMP system was associated with β 2-adrenergic receptors. The cAMP content was increased by isoproterenol (23-fold), a β-agonist, but not by octopamine, an α-agonist, and the affinity for ICI 118.551, a specific β 2-antagonist, was greater than for CGP 20712A, a specific β 1-antagonist (Kd: 0.03 × 10 −6 M and 15 × 10 −6 M respectively). The cAMP content was maximally increased by prostaglandin E 2 > β 2-adrenergic agonist > vasopressine type 2 receptor agonist (26-, 23-, and 3-fold the basal cAMP content, respectively). The present study demonstrates that cultured marginal cells retain some of their in vivo properties including a modulated enzymatic cAMP system. This culture model should allow further in-depth investigation of the function of marginal cells.

Acceleration of Retinol-Induced Epidermal Mucous Metaplasia by Stimulating the Dermal Adenylate Cyclase-cAMP System in Chick Embryonic Skin: Appearance of cAMP-Dependent Phosphorylated Proteins in Dermis of Retinol-Pretreated Skin after 2 h-Treatment with cAMP: (retinol/epidermal mucous metaplasia/dermis/adenylate cyclase/protein phosphorylation).

Epidermal mucous metaplasia of cultured 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing excess retinol (20 μM) for only 8-24 h and then in a chemically defined medium with Bt2 cAMP (0.2-2 mM) and without retinoids or serum for 2 days. In this work, stimulation of the adenylate cyclase-cAMP system in retinol-pretreated skin by forskolin, pertussis toxin, cholera toxin or AIF4 - was found to accelerate the synthesis of epidermal sulfated glycoprotein (mucin). In skin induced toward mucous metaplasia by retinol, treatment with forskolin for 1 day increased the cAMP content 10-fold in the dermis but only 2-fold in the epidermis over the control levels. The cAMP level of Bt2 cAMP (0.2 mM)-treated skin was 18 times higher in the dermis but rather lower in the epidermis than untreated skin. These results suggest the importance of an adenylate cyclase-cAMP system in the dermis of skin in stimulating mucous metaplasia induced by retinoids. In fact, cAMP-dependent protein phosphorylation was seen only in the dermis of retinol-pretreated skin after 2 h-treatment with cAMP. As no transfer of cAMP from the dermis to the epidermis of forskolin-treated skin was detected, there may be no gap junctional communication between the epidermis and the dermis, while the basement membrane becomes discontinuous during mucous metaplasia.

Intracellular mediation of GnRH action on GTH release in tilapia

The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala(6), Pro(9)-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca(2+) ionophore (A23187; 1-100 μM) or ionomycin (0.02-10 μM). The response of perifused pituitary to GnRH was reduced by nimodipine (1-10 μM) indicating that Ca(2+) influx via voltage-sensitive Ca(2+) channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16-160 μM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25-125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33-330 μM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 μM) in the presence of GnRH-antagonist ([Pro(2,6), Trp(3)]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca(2+) influx through voltage-sensitive Ca(2+) channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations.

Demonstration of inhibition of cyclic AMP accumulation in brain by very low concentrations of lithium in the presence of α-adrenoceptor blockade

In the present paper, we have studied the effect of lithium on cAMP levels induced by isoprenaline and norepinephrine in the presence of α- or β-adrenoceptor antagonists. Our results show that low lithium concentrations, starting at 0.3 × 10 −3 M, have a significant inhibitory effect on cAMP content induced by isoprenaline in brain tissue pretreated with the α-adrenoceptor blocker phenoxybenzamine. On the other hand, the inhibitory effect of lithium on cAMP levels induced by norepinephrine when β-adrenoceptors are blocked with propranolol, is observed at concentrations starting at 2.5 × 10 −3 M. These results show that in the presence of α blockade, low lithium concentrations which are within the therapeutic plasma range for treatment of manic patients, are role to act on an adenylate cyclase-cAMP system coupled to β-adrenoceptors.

Participation of the cytoskeleton in avian granulosa cell steroidogenesis

Cytochalasin B (CB) and the more specific cytochalasin D (CD), disruptors of microfilament polymerization, and colchicine, an inhibitor of microtubule polymerization, were studied for their effects on CAMP and steroid production in granulosa cells of domestic fowl. Each agent was incubated with freshly dispersed cells from the largest preovulatory follicle of laying hens taken 3–4 hr before expected ovulation. Total content (cells + medium) of cAMP and steroids was measured by established radioimmunoassays. CB dose dependently inhibited basal as well as LH- and forskolin-stimulated cAMP generation and diminished basal, LH- and 25-hydroxycholesterol (25-OHC)-supported progesterone production. Conversely, CD potentiated LH- and forskolin-promoted cAMP generation as well as LH- and 25-OHC-stimulated progesterone synthesis. Neither drug had any influence on metabolism of pregnenolone to progesterone. Colchicine had no effect on cyclic AMP generation, yet it suppressed progesterone synthesis by inhibiting the conversion of pregnenolone to progesterone. β-Lumicolchicine, a colchicine analog that does not depolymerize microtubules, had no such effect. The results suggest that microfilaments are involved in steroidogenesis at two sites, namely, the adenylate cyclase-cAMP system, and cholesterol conversion to pregnenolone; whereas microtubules act on the conversion of pregnenolone to progesterone.

Relaxation response of isolated canine veins to agents that act on the adenylate cyclase-cyclic AMP system: further investigation

The present study was undertaken to clarify whether there is a deficiency in the relaxation mediated by the adenylate cyclase-cyclic AMP (cAMP) system in the portal vein as compared to the saphenous vein. Longitudinal strips of the portal vein and helical preparations of the saphenous vein were used. The relaxation response to various agents was examined under conditions such that the venous preparations were previously contracted by methoxamine in equipotent concentrations (EC 80), i.e., 10 −6 M for portal vein and 10 −5 M for saphenous vein. The saphenous vein relaxed fully in response to isoproterenol but the portal vein relaxed only to 29% of the maximum relaxation induced by papaverine 10 −4 M. However, dibutyryl cAMP and 8-bromo-cAMP, membrane permeable derivatives of cAMP, 3-isobutyl-1-methylxanthine and papaverine, phosphodiesterase inhibitors, and forskolin, a direct stimulator of adenylate cyclase, relaxed portal and saphenous veins similarly though with quantitative differences. The results suggest that there is no profound deficiency in the adenylate cyclase-cAMP system but there may be a deficiency in the coupling between surface β-adrenoceptors and adenylate cyclase or there may be a low density of β-adrenoceptors in the portal vein.

Glucose-induced insulin secretion in uremia: Effects of aminophylline infusion and glucose loads

To explain mechanisms responsible for derangement of insulin release in uremia, we investigated glucose metabolism through three different tests in 14 patients with end-stage chronic renal failure. These tests were: intravenous glucose tolerance test with 0.33 g/kg of glucose solution (IVGTT); IVGTT with 0.5 g/kg of glucose solution (IVGTT2); IVGTT during aminophylline infusion (IVGTT + A). Twelve of the patients had IVGTT repeated after two to four months of thrice-weekly regular hemodialysis (IVGTT3). In each test we measured plasma glucose (G), immunoreactive insulin (IRI) and C-peptide. We also calculated glucose constant decay (K), insulin production (IRI area), insulinogenic index (IGI), and insulin resistance index (RI). Twenty-nine healthy volunteers formed the normal controls for IVGTT. As compared to controls, during IVGTT uremic patients showed significantly lower values in K, IRI area and IGI, and showed a significant RI value increase. During IVGTT2, IRI are values were higher than during IVGTT but IGI and K values were unchanged. During IVGTT + A both IRI area and IGI values were higher than during IVGTT. After hemodialysis treatment (IVGTT3) K, IRI areas and IGI increased significantly as compared to the predialysis period. K increase after hemodialysis correlated directly to IGI increase and inversely to RI changes. IGI increase during IVGTT3 was directly correlated to IGI rise during IVGTT + A. From these data we infer that defective insulin release in uremia is due to a decrease of beta-cell glucose sensitivity rather than to their functional exhaustion. An impaired adenyl cyclase-cAMP system may have an important role in the pathogenesis of this abnormality.

Interleukin-1-β Modulation of Prolactin Secretion from Rat Anterior Pituitary Cells: Involvement of Adenylate Cyclase Activity and Calcium Mobilization*

Recent findings indicate that interleukin-1 beta (IL1 beta), a monokine secreted by stimulated macrophages and monocytes, modulates neuroendocrine functions in a manner similar to classical hormones. In this study we show that IL1 modulates PRL secretion, assessed by reverse hemolytic plaque assay, and describe the effect of the monokine on adenylate cyclase activity and calcium fluxes in rat normal pituitary cells. In basal and vasoactive intestinal peptide (VIP)-stimulated conditions, low doses of IL1 reduced the mean plaque area, a direct index of PRL secretion without affecting the percentage of PRL-secreting cells. Similarly, low concentrations of IL1 inhibited adenylate cyclase activity in both basal and VIP-stimulated conditions, while higher concentrations restored the enzymatic activity to the control value. IL1 also caused a biphasic effect on the free intracellular calcium increase induced by maitotoxin, a calcium channel activator, being inhibitory at low and stimulatory at high concentrations. The effects of IL1 on adenylate cyclase activity and calcium fluxes were reversed by preincubation of the monokine with its polyclonal antibody, thus confirming the specificity of the effects. In conclusion, our data show that IL1 modulates PRL secretion by acting directly on pituitary cells through interaction with the adenylate cyclase-cAMP system and calcium flux.

D-2 Dopamine Receptor Activation Reduces Free [3H]Arachidonate Release Induced by Hypophysiotropic Peptides in Anterior Pituitary Cells*

Dopamine reduces the stimulation of intracellular [3H]arachidonate release produced by the two PRL-stimulating peptides angiotensin-II and TRH. This effect is concentration dependent and is mediated by stimulation of D-2 dopamine receptors. D-2 receptor agonists (bromocriptine, dihydroergocryptine, and dihydroergocristine) inhibit the release of fatty acid induced by angiotensin-II with a potency that parallels their ability to inhibit PRL release in vitro. Conversely, the selective D-2 receptor antagonist L-sulpiride completely prevents dopamine's effect, whereas SCH 23390 (a D-1 receptor antagonist) is ineffective. The inhibitory action of dopamine does not seem to be consequent to an action on the adenylate cyclase-cAMP system, as 8-bromo-cAMP (1 mM) does not affect either basal or dopamine-inhibited [3H]arachidonate release. However, a 24-h pertussis toxin pretreatment significantly reduces the action of dopamine on fatty acid release. Collectively, these results suggest that D-2 dopamine receptor-mediated inhibition of intracellular [3H]arachidonate release requires the action of a GTP-binding protein, but is not a consequence of an inhibitory action on cAMP levels.

Effect of follicle‐stimulating hormone on metabolism and maturation in Bufo arenarum oocytes

In the fully grown Bufo arenarum oocyte, carbohydrate breakdown during the autumn-winter season is accomplished mainly through the glycolytic pathway followed by the Krebs cycle. During the breeding season (spring-summer), carbohydrates are used mainly through the pentose phosphate cycle and through the variant of the Krebs cycle known as the glutamic aspartic cycle. The metabolism operating in the oocytes was determined using the following parameters: 1) the capacity of isolated mitochondria to oxidize citrate and fumarate; 2) the enzymatic activities of phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G-6-PDH); and 3) citrate and ATP compartmentalization. The present paper shows that follicle-stimulating hormone (FSH) would be one of the factors responsible for summer metabolism, since ovary fractions obtained from winter specimens treated with the hormone acquired the metabolic characteristics corresponding to oocytes obtained from breeding-season animals. From dose-response, and response in the function of time curves, it could be assumed that the optimum doses and times were 0.1 micrograms FSH/ml of incubation medium and 30 min treatment, respectively. The metabolic effect of FSH upon oocytes could be mediated by the adenylate cyclase-cAMP system, since treatment of ovaric fractions with cAMP 10(-3) M reproduced the effects obtained with the hormone. In addition, 0.02 mg/ml tetracycline proved to block the effect of FSH. A direct metabolic action of FSH on body cavity oocytes (without follicle cells) was observed when submitting these oocytes to the same hormonal treatment.

Effects of cold exposure on cyclic AMP concentration in plasma, liver, and brown and white adipose tissues in cold-acclimated rats

Effects of acute cold exposure on plasma energy substrates and tissue 3',5'-adenosine monophosphate (cAMP) were analyzed in intact rats, to define an involvement of the nucleotide in nonshivering thermogenesis (NST) and resultant cold acclimation. After an acute cold exposure to -5 degrees C, the plasma glucose level increased gradually in warm-kept control rats (C) while it decreased significantly in cold-acclimated rats (CA). However, it was increased considerably by an extreme cold exposure to -15 degrees C in both C and CA. By contrast, plasma levels of free fatty acids (FFA) increased immediately after cold exposure and the release lasted during the period of exposure especially in C. The cold exposure also increased plasma cAMP concentration but no concomitant increase was found in the liver. In both brown (IBAT) and white (WAT) adipose tissues the nucleotide concentration showed a stepwise decrease. The observed correlation between lipolysis and plasma cAMP response after cold exposure suggests an involvement of the adenylate cyclase-cAMP system in NST via lipid metabolism, at least, in the early stages of cold acclimation.

Effects of thyroid-stimulating hormone, carbachol, norepinephrine, and adenosine 3',5'-monophosphate on polyphosphatidylinositol phosphate hydrolysis in dog thyroid slices.

TSH (10-250 mU/ml), carbachol (100 nM to 10 microM), and norepinephrine (10-100 microM) stimulated in a dose-dependent manner the accumulation of [3H]glycerophosphoinositol, [3H]inositol monophosphate, [3H]inositol bisphosphate, and [3H]inositol triphosphate in dog thyroid slices prelabeled with myo-[2-3H]inositol. The maximal effect of carbachol was considerably greater than that of TSH and norepinephrine. Carbachol stimulation of [3H]inositol phosphates (InsPs) was present by 5 min of incubation, while that of TSH required at least 30 min. Neither the basal level of [3H]InsPs nor the stimulation by carbachol (1 microM) or norepinephrine (500 microM) was affected by forskolin (10 microM) or cAMP analogs [8-bromo-cAMP or (Bu)2-cAMP; 1 mM]. A maximal amount of TSH (250 mU/ml) had ergistic effects on submaximal carbachol (1 microM)- and additive effects on maximal norepinephrine (500 microM)-induced accumulation of [3H]InsPs. Since not all of the effects of TSH on the thyroid can be explained by activation of the adenylate cyclase system, these data indicate that TSH may also regulate thyroid function through the polyphosphatidylinositol phosphate pathway. In addition, stimulation of the adenylate cyclase-cAMP system does not modify the effects of carbachol or norepinephrine on [3H]InsPs accumulation in dog thyroid slices.

Studies on the Mechanism Responsible for Thyrotropin Induced Expression of Microsomal/Peroxidase Antigen in FRTL-5 Cells*

The expression of the microsomal (M) antigen on the surface and in the cytoplasm of a strain of rat thyroid cells (FRTL-5) is under the regulation of TSH. In the present report the mechanism by which TSH induces such expression was investigated with the use of human microsomal antibody-positive serum and an indirect immunofluorescence technique. Studies were also performed to ascertain whether the M antigen of FRTL-5 cells could be identified with thyroid peroxidase (TPO), as suggested by recent data obtained in human thyroid tissue. Preabsorption experiments showed that, like solubilized human thyroid microsomes, purified human TPO completely abolished the binding of microsomal antibody to FRTL-5 cells. No inhibition was obtained by preabsorption with control human tissues (placenta, liver, and spleen) or human thyroglobulin, indicating that the antigen recognized by microsomal antibody in FRTL-5 cells was TPO. After 72 h of TSH withdrawal from the culture medium the M/TPO antigen disappeared from the surface and the cytoplasm of FRTL-5 cells. Readdition of TSH (250 microU/ml) to the culture medium of cells lacking the M/TPO antigen elicited its reappearance within 24-48 h. This effect of TSH was prevented by 10 microM cycloheximide or 0.5-5 micrograms/ml actinomycin D. Two well known stimulators of the adenylate cyclase-cAMP system, cholera toxin and forskolin, mimicked TSH in inducing the reappearance of the M/TPO antigen. A similar effect was observed with use of the phosphodiesterase inhibitor isobutylmethylxanthine. Reappearance of M/TPO antigen was also produced by the cAMP analog 8-bromo-cAMP. The tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate, which stimulates thyroid cell growth through a cAMP-independent pathway, was ineffective in inducing the M/TPO antigen in FRTL-5 cells. The present data indicate that 1) thyroid peroxidase accounts for most, if not all, of the microsomal antigen of FRTL-5 cells; and 2) TSH modulates the expression of the M/TPO antigen in FRTL-5 cells by a mechanism that involves cAMP production and requires mRNA formation and subsequent protein synthesis.