How to localize parathyroid tumors in primary hyperparathyroidism?

The management of primary hyperparathyroidism (PHPT) has dramatically changed in the last 5 yr. Many more patients now undergo focused, limited or minimally invasive parathyroidectomy instead of traditional bilateral neck exploration. This change has taken place because of the improved accuracy of pre-operative localizing studies in selecting patients who have single-gland parathyroid disease (single adenoma) and can therefore have a minimally invasive parathyroidectomy. Sestamibi scanning followed by ultrasound, magnetic resonance imaging (MRI) and computed tomography (CT) scans are most accurate for localizing parathyroid tumors in patients with PHPT. Selective venous catheterization for PTH levels is useful when other localizing studies are negative or discordant in patients with persistent or recurrent PHPT. The routine use of one or more localizing studies commonly identifies the parathyroid tumor in patients with single-gland disease; but if localizing studies are negative or discordant, patients should have intra-operative PTH levels monitored or have a bilateral neck exploration to ensure a high rate of biochemical cure.

Expression of S100A2 and S100A6 in thyroid carcinomas.

AIMS: S100 calcium-binding proteins are known to play multiple roles in carcinoma development. In this study, we focused on two kinds of these proteins, S100A2 and S100A6, and investigated their expression in thyroid neoplasms. METHODS AND RESULTS: We investigated S100A2 and S100A6 expression in 141 thyroid neoplasms by immunohistochemistry. S100A2 was not expressed in normal follicles or follicular tumours, with one exception. Although 89.5% of papillary carcinoma were positive for S100A2, the expression was heterogeneous except in two cases. In anaplastic carcinoma, 78.5% of cases expressed S100A2 diffusely, while the remaining cases were negative. In normal follicles, S100A6 expression was always low, while 8.3% of follicular adenomas and 39.5% of follicular carcinomas showed increased expression. In papillary carcinomas, S100A6 expression was increased in 75% of cases, but in anaplastic carcinomas it was decreased, with only 14.3% showing high expression. CONCLUSIONS: The expression patterns of S100A2 and S100A6 in thyroid neoplasms are unique compared with those of other carcinomas, suggesting that: (i) S100A2 and S100A6 contribute to certain events in papillary carcinoma progression, and (ii) S100A2 expression is one of the biological characteristics of anaplastic carcinoma.

Tie-1 tyrosine kinase expression in human thyroid neoplasms.

AIMS: To investigate tie-1 expression in human thyroid neoplasms. Recent studies have demonstrated that receptor-type tyrosine kinases (RTKs) contribute to carcinoma progression. Tie-1 is one of the RTKs and plays a role in angiogenesis, although its pathophysiological significance in human carcinoma is still to be elucidated. METHODS AND RESULTS: Immunohistochemical expression of tie-1 was studied in various thyroid neoplasms. Tie-1 immunoreactivity was only occasionally observed in normal follicular cells. In papillary carcinoma, tie-1 was classified as positive in carcinoma cells in 55.7% of the cases and was more frequently expressed in those of smaller size with an absence of a poorly differentiated lesion. In contrast, tie-1 was positive in only 8.3% of anaplastic carcinoma and no cases of follicular carcinoma or adenoma were positive. CONCLUSIONS: These results suggest that tie-1 has a role in thyroid tumorigenesis, especially in the early phase of papillary carcinoma, but it is not important in the progression of anaplastic carcinoma or follicular tumour.

Polo-like kinase 1 overexpression is an early event in the progression of papillary carcinoma.

Polo-like kinase 1 (PLK1) is one of the serine threonine kinases that contributes to cell mitosis and is regarded as a marker of cellular proliferation. However, its protein expression in human carcinoma has not been studied in depth. We investigated PLK1 expression in various thyroid neoplasms in order to elucidate its physiological significance in thyroid carcinoma. Normal follicular cells only occasionally expressed PLK1. In follicular tumours and anaplastic carcinoma, PLK1 overexpression was not a common event and only 5.9% of follicular adenoma, 7.1% of follicular carcinoma, and 11.8% of anaplastic carcinoma overexpressed this protein. However, 43.7% of papillary carcinoma overexpressed PLK1. Polo-like kinase 1 overexpression was more frequently observed in smaller papillary carcinoma lesions, and 62.5% of microcarcinoma (ranging from 4 mm to 1.0 cm) and even 66.7% of incidental carcinoma (less than 4 mm) overexpressed it, whereas this phenomenon could only be seen in 20.0% of lesions larger than 4.0 cm. Furthermore, PLK1 overexpression was not related to cell-proliferating activity evaluated by Ki-67 labelling index, but it was inversely linked to UICC stage, extrathyroidal invasion, and the presence of poorly differentiated lesion as proposed by Sakamoto et al. These findings strongly suggest that, unlike other carcinomas previously studied, PLK1 does not act as a cell cycle regulator but plays a constitutive role in papillary carcinoma especially in the early phase, and may contribute to the malignant transformation of this carcinoma.

Activation of Arp2/3 complex-mediated actin polymerization by cortactin.

Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.

Distinct regulation of myoblast differentiation by intracellular and extracellular fibroblast growth factor-1.

We studied the role of fibroblast growth factor (FGF)-1 in the physiology of myoblast differentiation. We found that, while endogenous FGF-1 in L6-10 rat myoblasts did not suppress the progress of differentiation, the addition of FGF-1 to the culture medium suppressed it. Moreover, L6-10 cells stably transfected with full length FGF-1 undergo enhanced differentiation. The latter was well correlated with myogenin expression and myotube formation. Constitutive expression of a mutant FGF-1 (FGF-1U) that lacked a nuclear localization signal, promoted the differentiation of the myoblasts even more strongly. Furthermore, the expression of FGF-1U in an inducible expression system enhanced myogenin expression promptly. In L6-10 transfectants expressing a dominant-negative mutant of FGF receptor, stable transfection of FGF-1 promoted differentiation as it did in parent cells. Studies with FGF receptors and MAP kinase suggest that both are involved in the effect of FGF-1 when it is supplemented to culture medium but not during the effect of endogenous FGF-1 synthesized in cells. We conclude that intracellular (endogenous) and extracellular (exogenous) FGF-1 have differential effects on the regulation of myogenic differentiation of L6-10 cells.

Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin.

Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation. In the study reported here, we purified proteins that bind intracellular FGF-1. Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1. One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality. The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system. Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins. These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1.

The C-terminal region of fibroblast growth factor-1 is crucial for its biological activity and high level protein expression in mammalian cells.

We have studied the role of the carboxyl(C)-terminus of fibroblast growth factor(FGF)-1 using prokaryotic and eukaryotic expression systems. The full-length FGF-1 protein and its mutants lacking 6- and 9-amino acids at the C-terminus IFGF-1 (Cdel6) and FGF-1 (Cdel9), respectively] could be expressed in E. coli cells at the similar levels. The deletion mutants bound very weakly to FGF receptor and to heparin, and did not stimulate DNA synthesis in BALB/c3T3 cells. In contrast to E. coli cells, in NIH3T3 transfectants and L6 transfectants, the protein expression level of FGF-1 (Cdel6) was significantly lower than that of FGF-1, and longer C-terminal deletions further decreased the protein expression levels. However, the level of transcripts in the transfectants and the level of translates in in vitro system were equivalent for all the FGF-1 constructs. Treatment with proteasome inhibitors of the NIH3T3 transfectants expressing FGF-1(Cdel6) increased the protein level six-fold. The results indicate that the C-terminus of FGF-I is crucial for its biological activity and high-level expression in mammalian cells and suggest that deletion of the C-terminus of FGF-1 induces its post-translational degradation by proteasome system.

Expression of the fibroblast growth factor family and their receptor family genes during mouse brain development.

The fibroblast growth factor (FGF) family is composed of nine members and four genes encode protein tyrosine kinase receptors for them. To gain insight into the involvement of FGFs and their receptors in the development of nervous system, their expression in brains of perinatal and adult mice was examined by semi-quantitative reverse transcription-linked polymerase chain reactions and in situ hybridization. Although all the genes, with the exception of FGF-4, were found to be expressed, FGF-3, FGF-6, FGF-7 and FGF-8 genes demonstrated higher expression in the late embryonic stages than in postnatal stages, suggesting that these members are involved in the late stages of brain development. In contrast, expression of FGF-1 and FGF-5 increased after birth. Interestingly, FGF-6 expression in perinatal mice was restricted to the central nervous system and skeltal muscles, with intense signals in the developing cerebrum in embryos but in cerebellum in 5-day-old neonates. Furthermore, FGF-receptor (FGFR)-4, a cognate receptor for FGF-6, demonstrated similar spatiotemporal expression, suggesting that FGF-6 and FGFR-4 plays significant roles in the maturation of nervous system as a ligand-receptor system. The results indicate that individual member of the fibroblast growth factor and their receptor family are expressed either sequentially or simultaneously in brain development, strongly suggesting their involvement in the regulation of a variety of developmental processes of brain, i.e., proliferation and migration of neuronal progenitor cells, neuron and glia differentiation, neurite extensions, and synapse formations.

Further characterization of antagonism by alpha 2-adrenoceptor agonists of contractions induced by alpha 1-adrenoceptor agonists.

The population of alpha 1-adrenoceptor subtypes functionally mediating contraction in response to phenylephrine was examined in rat thoracic aorta and tail arteries. In thoracic aorta, chloroethylclonidine (CEC), which alkylates the alpha 1B and alpha 1D subtypes, shifted the concentration-response curve for phenylephrine substantially to the right without reduction in maximum contraction. The pA2 value (7.8) for 5-methylurapidil was consistent with the values reported for alpha 1D subtype, but was higher than those in tissues in which the alpha 1B subtype is dominant. In tail arteries, CEC did not shift the concentration-response curve for phenylephrine, but somewhat inhibited the maximum contraction. Schild analysis for 5-methylurapidil yielded a straight line with a slope significantly less than unity. Prazosin antagonized phenylephrine-induced contraction of tail arteries in a competitive manner with a pA2 value of 8.5, consistent with values for alpha 1L subtype. Clonidine relaxed the active tone induced by phenylephrine in both thoracic aorta and tail arteries, but quite different responses to clonidine by the two tissues were observed. After pretreatment with CEC, the relaxation induced by clonidine was abolished in thoracic aorta, but not in tail arteries. These results suggest that alpha 1D-and alpha 1L-adrenoceptors are mainly present in thoracic aorta and tail arteries, respectively. This difference in the populations of alpha 1-adrenoceptor subtypes may be related to regional differences in the modes of relaxant action of clonidine.

Characteristics of depression by clonidine of the pressor response to alpha 1-adrenoceptor agonists in pithed rats.

Effects of clonidine on the elevated arterial diastolic blood pressure induced by infusion of alpha 1-adrenoceptor agonists were studied in pithed rats. Intravenous injection of clonidine resulted in a dose-dependent pressor response at a resting state and a further increase in the moderately elevated diastolic blood pressure induced by infusion of lower doses of phenylephrine and methoxamine. However, clonidine produced a depressor response when diastolic blood pressure was elevated by around 100 mmHg during infusion of higher doses of alpha 1-adrenoceptor agonists. The depressor response to clonidine was dose-dependent at a dose range of 10 to 1000 micrograms/kg. On the other hand, clonidine failed to cause the depressor response while diastolic pressure was elevated by infusion of vasopressin by around 100 mmHg. NG-methyl-L-arginine, a nitric oxide synthase inhibitor, and propranolol, a beta-adrenoceptor antagonist, did not affect the depressor response to clonidine. After pretreatment with yohimbine, an alpha 2-adrenoceptor antagonist, the depressor response to clonidine was inhibited, but prazosin, an alpha 1-adrenoceptor antagonist, did not suppress the depressor response. These results demonstrate that clonidine specifically depresses the pressor response to alpha 1-adrenoceptor agonists in pithed rats via a mechanism which is not mediated by beta-adrenoceptors and independent of endothelium-dependent relaxing factor, and suggest that a yohimbine-sensitive mechanism may be related to the clonidine effect.

Inhibition of alpha1-adrenoceptor-mediated contractions in isolated tail arteries and aorta of the rat by alpha2-adrenoceptor agonists.

The effects of alpha2-adrenoceptor agonists, clonidine, tizanidine and UK-14304 on alpha1-adrenoceptor-mediated contractile responses were studied in isolated tail arteries and thoracic aorta of the rat. When applied during sustained contractile responses to almost maximum concentration (10 microM) of phenylephrine, clonidine (0.3 microM to 100 microM) produced concentration-dependent relaxations in both tissues. The maximum relaxation was smaller in tail arteries than in thoracic aorta. Clonidine up to 100 microM failed to relax both tissues precontracted with KCl (60 microM) or U-46619 (1 microM), a thromboxane mimetic. The clonidine-induced relaxation in tail arteries, was reversed by alpha2-adrenoceptor antagonists, yohimbine and idazoxane. Effects of the alpha2-adrenoceptor antagonists were concentration-dependent (0.1 microM to 1 microM), but not in a competitive manner. On the other hand, the relaxation in thoracic aorta was not significantly antagonized by these alpha2-adrenoceptor antagonists. Tizanidine and UK-14304 also relaxed both tail arteries and thoracic aorta precontracted with phenylephrine. The characteristics of the relaxation and their antagonism by yohimbine in both arteries were similar to those induce by clonidine. In tail arteries, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, at a concentration that completely inhibited acetylcholine-induced relaxations did not significantly affect the relaxation induced by clonidine. In contrast, the relaxation of thoracic aorta in response to clonidine was partly reduced in the presence of NG-nitro-L-arginine. These results indicate that the alpha2-adrenoceptor agonists selectively inhibit the contractions induced by phenylephrine in both tissues. Regional differences in the modes of the inhibition by the alpha2-adrenoceptor agonists exist.

Ameliorative effects of tea catechins on active oxygen-related nerve cell injuries.

Active oxygen species are suggested to be concerned with various senile disorders. Tea catechins, (+)catechin (CA), (-)epicatechin (EC) and (-)epigallocatechin gallate, are polyhydroxy-fravan derivatives from tea leaves and have been proposed to possess active oxygen scavenging effect. Tea catechins protected the cultured newborn-mouse cerebral nerve cells from death induced by glucose oxidase. The protective potency of (-)epigallocatechin gallate was weaker than those of EC and CA. Learning ability of mice was assessed by a step-down-type passive avoidance test, and memory impairment of mice was achieved by intracisternal injection of glucose oxidase or cerebral ischemia induced by 10 min occlusion of the common carotid arteries. Intracisternal injection of EC improved the memory impairment induced by intracisternal glucose oxidase, and i.v. injection of CA or EC improved that induced by the cerebral ischemia. CA and EC depressed carrageenin-induced edema in rat hind paw, but (-)epigallocatechin gallate did not. These results suggest that tea catechins ameliorate the injuries or impairments induced by active oxygens through scavenging intracellular active oxygens, and might become useful for protecting human from senile disorders such as dementia.

General pharmacology of the non-sulfhydryl angiotensin converting enzyme inhibitor N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline.

The effects of N-[8-amino-1(S)-carboxyoctyl]-L-alanyl-L-proline (AB-47, CAS 120008-53-9), an orally active angiotensin converting enzyme inhibitor, on the central nervous, respiratory and cardiovascular, autonomic systems, isolated smooth muscles and other functions were investigated in various experimental animals. AB-47 had no effect on central nervous, autonomic systems and isolated smooth muscles. AB-47 (10 and 30 micrograms/kg i.v.) significantly lowered femoral blood pressure without affecting respiration and heart rate in anesthetized rats. However, AB-47 had no effect on the contractile tension of mammalian isolated atrium and aorta. AB-47 had no effect on gastrointestinal transit in mice. Very slight injury of gastric mucosa was observed 4 h after the oral administration of AB-47 in rats but AB-47 did not damage the small intestinal mucosa. AB-47 had no effect on the contraction of rat phrenic nerve-diaphragm preparation induced by electrical stimulation. AB-47 did not affect the incidence of acetic acid-induces writhings. AB-47 potentiated carrageenan-induced hind paw edema in rats. The potentiation of edema may be due to an accumulation of bradykinin induced by the inhibition of angiotensin converting enzyme (ACE), because ACE is the identical enzyme with kinase II. The pretreatment of AB-47 for 7 days (1, 3, 10 mg/kg/d p.o.) inhibited the cardiac hypertrophy induced by isoproterenol (isoprenaline). This result suggests that the renin-angiotensin-aldosterone system directly or indirectly participates in the cardiac hypertrophy induced by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of antinociception induced by noncatecholic phenylethylamine derivatives: the involvement of alpha-2-adrenoceptors.

Characteristics of the antinociceptive action of phenylethylamine derivatives, amphetamine, beta-phenylethylamine (PEA) and beta-hydroxyphenylethylamine (OHPEA), were examined. The antinociception induced by PEA derivatives was enhanced by intracisternal injection of norepinephrine or clonidine and attenuated by intracisternal injection of phentolamine or yohimbine, but was not affected by intracisternal injection of prazosin in the mouse hot plate method. PEA derivatives induced a contraction of the rat vas deferens, and this contraction by PEA derivatives was attenuated by the application of phentolamine. The contractions induced by PEA or OHPEA in the reserpinized vas deferens were much smaller than those in the normal one. PEA derivatives inhibited the electrical stimulation-evoked contractions of the vas deferens, and the inhibition by PEA derivatives was reversed by the application of yohimbine. These findings indicate that PEA derivatives may induce the antinociception as a result of stimulating the alpha 2-adrenoceptors. The stimulation of alpha 2-adrenoceptors by PEA derivatives may result from the release of endogenous norepinephrine and/or from direct action on the alpha 2-adrenoceptors.

Possible mechanisms of action of the antispasmodic agent tiropramide in the isolated detrusor from rats.

The effects of tiropramide hydrochloride on Ca(2+)-induced contraction, cytoplasmic free Ca2+ levels and tissue cyclic AMP concentrations were investigated to elucidate the mechanisms of its antispasmodic action in the isolated detrusor from rats. Tiropramide inhibited the Ca2+ (3 mM)-induced contractions of the isolated urinary bladder depolarized in a Ca(2+)-free medium, and the IC50 value was 3.3 x 10(-6) M. When tiropramide was added during the sustained phase of the K+ (60 mM)-contracture, IC50 values of tiropramide for the contraction and the increased fluorescence were 1.9 x 10(-5) M and 16.4 x 10(-5) M, respectively. On the other hand, the IC50 values for the K(+)-induced contraction and fluorescence after pretreatment of the isolated urinary bladder with tiropramide were 2.1 x 10(-5) M and 2.6 x 10(-5) M, respectively. Tissue cyclic AMP levels at 1 min after addition of 10(-5) M tiropramide were significantly increased. Papaverine, IBMX or forskolin potentiated the inhibitory effect of tiropramide on carbachol-induced contraction and its cyclic AMP-elevating effect. However, a good correlation between the degrees of potentiation of the inhibitory effect and the increase in cyclic AMP levels was not observed. The present results suggest that the smooth muscle relaxant activity of tiropramide in the isolated detrusor from rats may be intimately associated with predominant inhibition of Ca2+ influx and, to a lesser extent, an increase in intracellular cyclic AMP levels.

[Effects of tiropramide hydrochloride on the isolated detrusor and intravesical pressure of the bladder in situ in rats].

The effects of tiropramide on the isolated detrusor and intravesical pressure of the bladder in situ in rats were compared with those of flavoxate, oxybutynin and terodiline. The IC50 values (x 10(-5) M) of tiropramide for carbachol (CCh)-, K+ (60 mM)-, Ba2+ (10 mM)-, and electrical stimulation-induced contractions were 3.6, 4.2, 5.8, and 2.9, respectively. The four antispasmodics used (2 and 4 mg/kg, i.v., each) abolished the rhythmic bladder contractions in situ in anesthesized rats. Of the four compounds, oxybutynin was most potent and no significant differences were observed between the inhibitory effects of tiropramide, flavoxate and terodiline. The administration of flavoxate (30 and 60 mg/kg) into the duodenum little influenced the rhythmic bladder contractions. Tiropramide, flavoxate, oxybutynin and terodiline (8 and 12 mg/kg, i.v., each) dose-dependently prolonged the time to the volume-evoked micturition reflex, and the activity of tiropramide was not statistically different from those of the other three antispasmodics. Under unilateral pelvic and bilateral hypogastric nerve transection, both of the contractions induced by electrical stimulation of the peripheral and central cut ends of the pelvic nerve were dose-dependently inhibited to the same extent by tiropramide and terodiline. These results suggest that the effects of tiropramide on the function of urinary bladder in rats may be mainly due to direct actions on the smooth muscle, and that tiropramide is more potent than flavoxate and less potent than oxybutynin and terodiline.