Interleukin 4 as an essential factor for in vitro clonal growth of murine connective tissue-type mast cells.

We investigated the biological activity of IL-4 to murine connective tissue-type mast cells (CTMC). When purified peritoneal mast cells, typical CTMC, were incubated with pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) in methylcellulose, about one-fifth of mast cells showed clonal growth. Recombinant IL-4 alone did not stimulate the clonal growth, and purified IL-3 alone induced development of a small number of tiny clusters. In contrast, addition of IL-4 to IL-3 increased the number of clusters by a factor of 10. The number and size of clusters induced by the combination of IL-3 and IL-4 were comparable to those of mast cell clusters induced by PWM-SCM. The present results indicate that IL-4 is an essential factor for in vitro clonal growth of CTMC.

Accumulation of apolipoprotein E-rich high density lipoproteins in hyperalphalipoproteinemic human subjects with plasma cholesteryl ester transfer protein deficiency.

This study characterized the plasma lipoproteins of familial hyperalphalipoproteinemic patients with or without deficiency of cholesteryl ester transfer protein (CETP) activity. The subjects with CETP deficiency have increased levels of apolipoprotein (apo) E. The increased concentration of apo E in these subjects was correlated to the appearance of apo E-rich high density lipoproteins (HDL). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed that these lipoproteins contained predominantly the apo E (82%) and little amount of apo A-I (18%). These apo E-rich HDL displayed a much higher affinity than human LDL in binding to LDL receptors on human fibroblasts. Furthermore, 3.5 times fewer apo E-rich HDL than LDL were required to saturate the receptors on fibroblasts. These data indicated that the apo E-rich HDL in CETP-deficient human subjects contained multiple copies of apo E and bound to the LDL receptor through multiple interactions. The apo E-rich HDL, with similar properties as cholesterol-induced apo E HDLc, were not detectable in normal human subjects or in hyperalphalipoproteinemic subjects with normal CETP activity. The apo E-containing HDL in the latter subjects were smaller and contained only small amounts of apo E (14%). The difference in apo E-containing HDL in these subjects suggests a correlation between CETP level and the appearance of apo E-rich HDL.

Excess purine degradation in exercising muscles of patients with glycogen storage disease types V and VII.

To investigate purine catabolism in exercising muscles of patients with muscle glycogen storage disease, we performed ischemic forearm exercise tests and quantitated metabolites appearing in cubital venous blood. Two patients with glycogen storage disease type V and three with glycogen storage disease type VII participated in this study. Basal lactate concentrations lowered in every patient with glycogen storage disease type V or type VII. Two patients with glycogen storage disease type VII, who had markedly elevated concentrations of serum uric acid (14.3 and 11.9 mg/dl, respectively), showed high basal concentrations of ammonia (118 and 79 mumol/liter, respectively; 23 +/- 4 mumol/liter in healthy controls) and of hypoxanthine (23.4 and 20.4 mumol/liter, respectively; 2.0 +/- 0.4 mumol/liter in healthy controls). Other patients showed near normal measurements of these metabolites. After forearm exercise, ammonia, inosine, and hypoxanthine levels increased greatly in every patient studied, in contrast with the lack of increase in lactate levels. The incremental area under the concentration curves for venous ammonia was 13-fold greater in the glycogen storage disease group than in controls (1,120 +/- 182 vs. 83 +/- 26 mumol X min/liter). The incremental areas of inosine and hypoxanthine were also greater in the glycogen storage disease group (29.2 +/- 7.2 vs. 0.4 +/- 0.1 and 134.6 +/- 23.1 vs. 14.9 +/- 3.2 mumol X min/liter, respectively). The incremental areas of ammonia in controls and in glycogen storage disease patients strongly correlated with those of hypoxanthine (r = 0.984, n = 11, P less than 0.005). These findings indicated that excess purine degradation occurred in the exercising muscles of patients with glycogen storage disease types V and VII, and suggested that the ATP pool in the exercising muscles may be deranged because of defective glycogenolysis or glycolysis.

Properties of glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with familial cortisol resistance.

In a previous report of two patients with familial glucocorticoid resistance due to reduced numbers of glucocorticoid receptors (GR), we have shown decreased numbers of GR in peripheral mononuclear cells and cultured fibroblasts but normal affinity of GR in both patients. In this study, peripheral lymphocytes from these patients, one patient's son and daughter, and normal subjects were transformed with Epstein-Barr virus. Reduced numbers and normal affinity of GR were found in the Epstein-Barr virus-transformed lymphocytes from both patients while the son and daughter had normal numbers and affinity of GR. The thermal stability of GR and thermal activation of cytosolic receptors in both patients were found to be normal. Although the percentages of nuclear bound GR were similar in both patients and normal controls, the absolute amounts of nuclear bound GR of the patients were about one-half that of normal controls. These abnormal properties of GR (reduced numbers of GR) were preserved in the transformed cells from the patients.

Increase in the active form of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human hepatocellular carcinoma: possible mechanism for alteration of cholesterol biosynthesis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity and the rate of sterol biosynthesis are positively correlated with DNA synthesis and proliferation of mammalian cells. The total (active plus latent) activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and the activity of its active form in hepatocellular carcinoma (HCC) from seven patients were measured and compared with those in liver tissue from five control subjects. The activity of the active form in HCC was 61 +/- 21 (SD) pmol/min/mg microsomal protein, while it was only 17 +/- 9.8 pmol/min/mg protein in the liver tissue from the controls; the difference was significant (P less than 0.005). The total activity of the reductase was also higher in HCC although the difference was not significant. The microsomal contents of the enzyme protein also were not significantly different. The rate of cholesterol biosynthesis was 307 +/- 81 pmol/h/mg tissue in HCC and 79.6 +/- 52 in normal liver tissue, indicating a significant increase in the rate in HCC (P less than 0.001). Thus, enhanced synthesis of cholesterol in human HCC seems to result partly from an increase in the active form of the reductase.

Elevated levels of transforming growth factor beta messenger RNA and its polypeptide in human hepatocellular carcinoma.

Malignant cells in culture express elevated levels of transforming growth factor beta 1 (TGF-beta 1) mRNA and secrete an abundant amount of TGF-beta protein, but little is known about the production of TGF-beta in human malignant tissues in vivo. We estimated the levels of TGF-beta 1 mRNA expression by Northern hybridization and measured TGF-beta protein using a radioreceptor assay in tumor tissues surgically obtained from six patients with hepatocellular carcinoma (HCC). TGF-beta 1 mRNA was expressed at much higher levels in HCC tissues from all the cases compared with normal human liver, suggesting an association of the activated TGF-beta 1 gene transcription with hepatocarcinogenesis. The content of TGF-beta was 207 +/- 121 ng/g wet tissue in the HCC tissue, and it showed correlation with the level of TGF-beta 1 mRNA in the tissue (r = 0.69; P less than 0.05). An immunohistochemical study demonstrated that TGF-beta 1 staining could be observed in HCC cells. These observations suggest that human HCC strongly expresses TGF-beta 1 mRNA in vivo, leading to a high content of TGF-beta protein.

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver.

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b5, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b5 than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450PB purified from rabbit liver, addition of cytochrome b5 to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b5 enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.

Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa).

To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa). In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues. Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold). The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues. There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats. LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold). The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold). Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity.

Alteration of glycolytic intermediary metabolism in erythrocytes during diabetic ketoacidosis and its recovery phase.

The concentrations of glycolytic intermediates and adenine nucleotides were determined in erythrocytes from patients with diabetic ketoacidosis before and during insulin treatment. Ketoacidosis resulted in an increase in the levels of intermediates above the phosphofructokinase (PFK) step and a marked decrease in the levels of those below this step. Thus, a "crossover" point was seen at the PFK step in a crossover plot. This indicated that the rate of glycolytic flow during ketoacidosis was controlled by PFK and that the reduced level of 2,3-bisphosphoglycerate (2, 3-BPG) was attributed to the inhibition of this enzyme. In vitro studies revealed that acidemia is mainly responsible for the inhibition of PFK, whereas elevated levels of ketone bodies and free fatty acids have no direct bearing on it. Insulin administration produced hypophosphatemia within 8-12 h and it persisted for 24 h or longer. The levels of fructose-6-phosphate and glucose-6-phosphate were decreased transiently during this hypophosphatemic phase, while those of fructose bisphosphate and triose phosphates were increased. This indicated that PFK was activated. Thus, it is no longer reasonable to think that the inhibition of PFK is a factor responsible for a delay in normalization of the 2, 3-BPG level during the recovery phase. The levels of these glycolytic intermediates, including 2, 3-BPG, were normalized within 4 days by appropriate therapy.

Preventive and therapeutic effects of large-dose nicotinamide injections on diabetes associated with insulitis. An observation in nonobese diabetic (NOD) mice.

This experiment was undertaken to explore a novel method of therapy for insulin-dependent diabetes mellitus (IDDM), using nonobese diabetic (NOD) mice that had symptoms and histologic changes similar to those of human IDDM patients. We examined preventive and therapeutic effects of large-dose nicotinamide administration on diabetes in NOD mice. Eighteen young female NOD mice without glycosuria were randomly divided into two groups; nine received subcutaneous nicotinamide (0.5 mg/g body wt) injections every day and the other nine were maintained as a control group and not injected. After 40 days, all of the mice given nicotinamide showed almost normal glucose tolerance and only mild insulitis on histologic study. On the other hand, marked glycosuria and severe insulitis were observed in six of the nine mice not injected. Four of six NOD mice given nicotinamide from the day of the first occurrence of marked glycosuria displayed a disappearance of glycosuria and an improvement in glucose tolerance during the therapy; however, urine sugar became negative in only one of six mice that received nicotinamide from 1 to 2 wk after the onset of marked glycosuria. These results indicate that nicotinamide has preventive and therapeutic effects on diabetes in NOD mice, and suggest the reversibility of B-cell damage, at least at a very early stage of IDDM.

Mechanism of hypoglycemia observed in a patient with insulin autoimmune syndrome.

A 21-year-old female patient complaining of frequent hypoglycemic attacks in the presence of a large amount of circulating insulin-binding antibodies without previous known immunization is described. In order to clarify the possible mechanism of the hypoglycemic attacks occurring in this new syndrome, changes in plasma glucose, plasma total and free immunoreactive insulin (IRI), and C peptide immunoreactivity (CPR) levels were investigated in the patient before, during, and after a three-hour glucose infusion. The character of her antibodies were also examined. An abrupt discontinuation of the glucose infusion caused a sharp decline in the plasma glucose level, reaching a nadir of 30 mg./100 nk, at 270 minutes; then she became unconscious. A huge amount of total IRI of 2,834 micron U./ml. was registered at 180 minutes, while the peak value of free IRI of 208 micronU./ml. was observed 45 minutes after the cessation of the glucose infusion. Plasma CPR was increased from high basal level, 19.6 ng./ml., to the maximum level of 29.2 ng./ml. The maximum insulin-binding capacity of IgG in the patient's serum was 6.25 mU./ml. The antibody-combining site was homogeneous, showing one high-affinity site (K: 1.1 X 10(9)M-1). Neither the prolonged fasting nor the administration of tolbutamide induced the hypoglycemic attack in the patient. The hypoglycemia may be explained by an unduly excessive amount of insulin liberated from a large pool of bound insulin irrespective of blood sugar level. The cause of the antibody production is also discussed.

Increased activity of intestinal acyl-CoA: cholesterol acyltransferase in rats with streptozocin-induced diabetes and restoration by insulin supplementation.

We examined the activities of intestinal acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol esterase, enzymes regulating cholesterol absorption, in rats with streptozocin-induced diabetes (STZ-D) to clarify the effect of diabetes on cholesterol absorption. Three weeks after the induction of diabetes, plasma cholesterol levels were slightly but significantly increased in diabetic rats compared with control animals, whereas a far more remarkable increase in plasma cholesterol was observed in diabetic rats when fed an atherogenic diet containing 1% cholesterol, 0.5% cholic acid, and 5% lard. Microsomal ACAT activity in intestinal mucosa was three times higher in diabetic than in control rats. However, no significant difference in the enzyme activity could be detected between diabetic animals fed control chow and those fed the atherogenic diet. Furthermore, insulin supplementation given to diabetic rats caused a reduction of enzyme activity to the levels found in control animals. In contrast, cholesterol esterase activity in rat intestinal mucosa was unaffected by either the induction of diabetes or the atherogenic diet feeding. In conclusion, we disclosed that apparent ACAT activity in intestinal mucosa is elevated in STZ-D rats. Therefore, we postulate that enhancement of CoA-dependent cholesterol esterification in the intestine might be one of the major factors responsible for hypercholesterolemia in diabetes.

Glucagonostatic and insulinotropic action of glucagonlike peptide I-(7-36)-amide.

We examined the effect of glucagonlike peptides (GLPs), which are cleaved from preproglucagon in the enteroglucagon cells, on rat endocrine pancreas with the isolated perfused system. GLP-I-(7-36)-amide, a truncated form of full-sequence GLP-I-(1-37), showed a potent inhibitory effect on glucagon secretion. This inhibitory effect of GLP-I-(7-36)-amide was demonstrated at concentrations of 0.25, 2.5, and 25 nM in 11.2 and 2.8 mM glucose. In contrast, insulin release was significantly stimulated by GLP-I-(7-36)-amide at its concentration from 0.025 to 25 nM in a high glucose concentration, whereas in a low glucose concentration, the stimulation was seen only at the highest concentration (25 nM). Neither GLP-I-(1-37) nor GLP-II showed any effect on glucagon and insulin release. Although several gastrointestinal hormones have been nominated as incretins, none of them may suppress the glucagon secretion. A truncated form of GLP-I, GLP-I-(7-36)-amide thus seems to be a unique incretin that exerts glucagonostatic action.

Demonstration of islet cell surface antibodies in sera of New Zealand black mice and inhibitory effect on insulin release.

Islet cell surface antibodies (ICSAs) in sera of New Zealand Black (NZB) and New Zealand White (NZW) mice were detected by the indirect immunofluorescence method with cultured Balb/c mouse islet cells as antigens. Circulating ICSAs appeared in NZB mice from age 20 wk; at 30 wk, 73% of male mice and 88% of female mice had detectable ICSAs. The ICSAs were significantly absorbed with mouse islet cells but hardly absorbed with spleen cells or liver powder. The ICSAs also bound with islet cells of ICR mice, Sprague-Dawley rats, and NZB mice. NZB mice showed glucose intolerance especially at ages 10 and 30 wk. Although plasma glucose levels tended to be higher in NZB mice with strongly positive ICSAs, pancreatic insulin content was not reduced, and insulitis was rarely observed in the pancreases. On the other hand, 30-wk-old NZW mice had normal or mildly impaired glucose tolerance and only weak, if any, ICSAs. The ICSA-positive serum of NZB mice significantly suppressed glucose-induced insulin release by cultured islet cells. The ICSAs may be responsible, at least in part, for glucose intolerance in NZB mice after age 20 wk through the inhibitory effect on insulin secretion.

Clinical pharmacology of human insulin of recombinant DNA origin in healthy volunteers.

The serum insulin, fractional absorption, serum human C-peptide, and plasma glucose responses of normal fasting subjects were compared after the subcutaneous and intravenous administration of human insulin (recombinant DNA) and Novo Actrapid insulin. While no statistical difference at each time point was observed between the two insulins, for each time point after 0.05 and 0.1 U/kg s.c., the 0-6 h area under curve (AUC) after the 0.05 U/kg dose was greater for human insulin than for pork insulin. There was no difference in the 0-6 h AUC after the 0.1 U/kg s.c. dose. The serum human C-peptide responses to the two insulins were virtually identical. With the 0.05 U/kg s.c. dose, hypoglycemic effect of human insulin was greater than for Actrapid. This difference did not occur after 0.1 U/kg s.c. Following intravenous administration using 0.05 U/kg, the serum IRI, serum C-peptide, and glucose responses were the same. These data indicate only slight differences between human insulin and Actrapid insulin.

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts.

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

Inhibitory effect of iopanoic acid on the thyrotropin-stimulated release of cyclic adenosine 3',5'-monophosphate and of 3,5,3'-triiodothyronine from perifused rat thyroids.

Using a perifusion system, we studied the effect of iopanoic acid, an iodinated contrast agent used in oral cholecystography, on the release of cAMP, T3, T4, and rT3 from perifused rat thyroid pieces. A 0.1 mg/ml iopanoic acid solution significantly inhibited the TSH-stimulated release of cAMP (without iopanoic acid, 8175 +/- 373; with iopanoic acid, 5169 +/- 355 fmol/mg thyroid X 3 h, mean +/- SE) and T3 (without iopanoic acid, 971 +/- 32; with iopanoic acid, 659 +/- 32 pg/mg thyroid X 3 h) in the presence of 3-isobutyl-1-methylxanthine. T4 and rT3 releases were not significantly affected. Inhibition of TSH-stimulated T3 release by iopanoic acid was also observed at a concentration of 0.01 mg/ml. Propylthiouracil completely abolished the inhibitory effect of iopanoic acid on TSH-stimulated cAMP release but not on TSH-stimulated T3 release. TSH-stimulated cAMP release was augmented by iodide at a concentration of 1 X 10(-3) M in the presence of 3-isobutyl-1-methylxanthine but suppressed by iodide at a concentration of 1 X 10(-5) M. TSH-stimulated T3 release was suppressed slightly at both concentrations of iodide. These results suggest that iopanoic acid may have an inhibitory effect on the TSH-stimulated cAMP and T3 release from perifused rat thyroids. This effect can probably be attributed to the iodide contained in the agent and to the inhibited intrathyroidal conversion of T4 to T3.

Effect of an exposure to chloramine-T on the immunoreactivity of glucagon.

Various preparations of glucagon treated with chloramine-T under different conditions have been studied with respect to their immunoreactivity toward two different glucagon antisera; one specific for pancreatic glucagon and the other capable of reacting with enteroglucagon as well. The glucagon preparations exposed to chloramine-T for different periods reacted almost identically with the nonspecific antibody whether they were used as tracer or standard. On the contrary, treatment with chloramine-T under severe conditions led to reduced immunoreactivity toward the specific antibody. Inclusion of dimethyl sulfoxide (DMSO) in the chloramine-T reaction resulted in preservation of the immunoreactivity of the treated preparations. The cyanogen bromide cleaved-glucagon, (1-26) homoserine lactone, showed little cross-reactivity with the specific antibody whereas it reacted to a similar extent with the nonspecific antibody as natural glucagon did. Amino acid analysis of the hormone exposed to chloramine-T demonstrated that the methionine residue at position 27 in the glucagon molecule had been oxidized to methionine sulfoxide. In addition, tryptophan had also been affected. DMSO protected methionine and tryptophan from the oxidative action of chloramine-T. We postulate from these results that the change in the immunoreactivity toward the specific antibody of glucagon exposed to chloramine-T is mainly due to oxidation of the methionine residue at position 27 in the molecule. The usefulness of DMSO in the iodination process is also discussed.

Inhibitory effect of lithium on the release of thyroid hormones from thyrotropin-stimulated mouse thyroids in a perifusion system.

We studied the effect of lithium on the release of T3, T4, and cAMP from perifused mouse thyroids and on cAMP content in thyroid pieces. Lithium significantly inhibited T3 and T4 release from TSH-stimulated mouse thyroids. This inhibitory effect on thyroid hormone release was dependent on the concentration of lithium. Under continuous stimulation with TSH and 3-isobutyl-1-methylxanthine, both cAMP release and cAMP content were significantly decreased by lithium. In addition, we studied the effect of lithium on (Bu)2cAMP-stimulated thyroid hormone release. T3 and T4 release was stimulated by (Bu)2cAMP in a similar way to TSH. Lithium significantly inhibited (Bu)2 cAMP-stimulated T3 and T4 release from perifused mouse thyroids. These results suggest that lithium inhibits the action of TSH in the thyroid gland by both suppression of cAMP production and inhibition at a step beyond cAMP generation.

Effects of iodide and propylthiouracil on the release of 3,5,3'-triiodothyronine and of cyclic adenosine 3',5'-monophosphate from perifused rat thyroids.

We have established a perifusion system using rat thyroid. With this system, we analyzed the effects of TSH, 3-isobutyl-1-methylxanthine (IBMX) and iodide on the release of T3 and of cAMP, paying special attention to the relation between the release of the two substances. During perifusion, which was continued for 7 h, T3 release increased progressively with time and in a dose-related manner when TSH was added at concentrations of 0.1-10 mU/ml. However, cAMP release was unappreciable even in the presence of 10 mU/ml TSH. The release of T3 and cAMP was markedly enhanced by 3 x 10(-4) M IBMX. When iodide was added, a marked increase in cAMP release was unexpectedly observed. However, a slight but significant suppression of TSH-stimulated T3 release was shown with 1 x 10(-3) M iodide. TSH-stimulated T3 release was almost completely inhibited by 1 x 10(-3) M 6-n-propyl-2-thiouracil, but such a complete inhibition did not occur with 2-mercapto-1-methylimidazole. The cAMP release stimulated by IBMX was not affected by 6-n-propyl-2-thiouracil, but that stimulated by iodide was effectively inhibited. The present studies indicate that TSH and IBMX enhance T3 release, but only IBMX increases cAMP release. Iodide also results in a marked increase in cAMP release but does not affect T3 release from unstimulated thyroid and inhibits T3 release from TSH-stimulated thyroid. We suggest that there is not necessarily any close correlation between T3 release and cAMP release into perifusates of the rat thyroid.