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Anticancer drug-induced stomatitis develops in 30% to 40% of cancer patients undergoing chemotherapy. However, medications for this condition are not commercially available in Japan. The "hospital formulation" is a customized medicine which hospital pharmacists prepare when doctors cannot carry out the medical therapy most suitable for a patient using commercial medicines. However, as the duties of pharmacists increase, use of the "hospital fomulation" decreases. Therefore, development of "hospital fomulations" based on individual evidence has a limit. Irsogladine maleate (IM) is a drug with gastric mucosal protective properties. IM increases intracellular cAMP levels in the gastric mucosa and activates communication between cells. It has been reported that the oral administration of IM reduces the incidence of 5-FU-based chemotherapy-induced stomatitis. However, there have been no reports on the effect of the direct use of IM in treating stomatitis. Therefore, we studied the development of an IM oral spray for stomatitis treatment, and obtained evidence of a direct effect in an animal experiment using a stomatitis model. Next, rebamipide mouthwash was administered to patients who had stomatitis caused by cancer chemotherapy. The total scores were classified into Grades 0 to 4 and evaluated as a stomatitis evaluation score (SES). When comparing SES and changes in the stomatitis area in patients, gradual reductions in the extent of stomatitis were observed, even during the period when SES did not change. Having patients fill in an observation chart was effective for grasping changes in symptoms in outpatients.

Upon elevation of plasma glucose concentration, pancreatic β-cells generate bursts of action potentials to induce cyclic changes in [Ca(2+)]i regulating insulin release. Glucose-dependent insulin secretion is synergistically enhanced by glucagon-like peptide-1 (GLP-1), which increases [cAMP]i and activates protein kinase A (PKA) and exchange protein activated by cAMP (Epac). The insulinotropic effect of GLP-1 is mediated, at least in part, by modulating multiple ion channels/transporters at the plasma membrane and ER through PKA- and EPAC-dependent mechanisms, which increase membrane excitability and intracellular Ca(2+) release. However, because of complex interactions between multiple cellular factors involved in the GLP-1 effects, quantitative aspects of the molecular/ionic mechanisms have not yet been determined. We thus performed simulation studies and mathematical analysis to investigate how GLP-1 signals control [cAMP]i and subsequently modify the bursting activities and Ca(2+) dynamics. First, a GLP-1 receptor signal transduction model was developed and introduced to our β-cells model. Secondly, modulatory effects of PKA/Epac on ion channels/transporters were incorporated based on experimental studies. Increases in the frequency and duration of the bursting activity observed during GLP-1 stimulation were well reconstructed by our model, and lead potential analysis quantitatively determined the functional role of each ion channel/transporter in modifying the burst pattern. Finally, an IP3R model was developed to reproduce GLP-1-induced Ca(2+) transients/oscillations. Instantaneous equilibrium analysis and bifurcation analysis also elucidated the quantitative mechanisms involved in generating IP3R-mediated Ca(2+) mobilization. The results of this theoretical analysis of the effects of GLP-1 on membrane excitability/Ca(2+) dynamics are discussed in this review.

Although the central nervous system and the neurotransmitters are known to control not only the immune system but also the homeostasis of bone mass, their pathological relevance to bone disorders remains unclear. Osteoclasts in the synovium of rheumatoid arthritis (RA) play an important role in bone destruction. It is known that increased sympathetic nervous activity increases both differentiation and function of osteoclasts, which leads to bone loss. Dopamine, a major neurotransmitter, transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. We previously reported that dopamine plays an important role in IL-6-IL-17 axis and subsequent joint destruction in RA. The major source of dopamine in the synovial tissue of RA was dendritic cells (DCs) that stored and secreted dopamine. Dopamine released by DCs bounded to D1-like dopamine receptors on T cells and induced activation of cAMP and differentiation to Th17 cells via IL-6 production We here overview the interplay among the immune system, bone metabolism and neurologic system shedding light upon dopaminergic signals upon osteoclastogenesis.

Cilostazol, a selective inhibitor of phosphodiesterase III, is an antiplatelet drug and a vasodilator via increased cAMP levels. It has been approved for the treatment of ischemic symptoms in chronic peripheral arterial obstruction or intermittent claudication and for secondary prevention of cerebral infarction (CSPS I). Recently, cilostazol has been reported to be more effective than aspirin in the secondary prevention of all types of stroke in patients and, in particular, prevent the secondary attack of hemorrhagic stroke in patients (CSPS II). Laboratory investigations revealed that cilostazol has a neuroprotective effect against ischemic brain injury. The neuroprotective potential is dependent on its antiinflammatory and antiapoptotic effects mediated by scavenging hydroxyl radicals, decreasing formation of tumor necrosis factor-α, and inhibition of poly (ADP-ribose) polymerase activity. In addition, increasing evidence indicates that cilostazol may offer endothelial protection via both the inhibition of lipopolysaccharide-induced apoptosis and induced nitric oxide (NO) production by endothelial NO synthase activation. The breakdown of the barrier permeability of the blood brain barrier (BBB) often accelerates the progression of diseases such as cerebral ischemia. However, the molecular mechanisms involved in BBB disruption have not been fully determined. Identification of the molecules responsible for the disruption of the endothelial barrier may yield new therapeutic targets in intractable diseases. This article reviews the protective effects of cilostazol against transient focal cerebral ischemia and hemorrhagic transformation and its mechanism of action.

The first step in olfactory sensation involves the binding of odorant molecules to specific receptor proteins on the ciliary surface of olfactory receptor cells (ORCs). Odorant receptors coupled to G-proteins activate adenylyl cyclase leading to the generation of cAMP, which directly gates a cyclic nucleotide-gated cationic channel in the ciliary membrane. This initial excitation causes a slow and graded depolarizing voltage change, which is encoded into a train of action potentials. Action potentials of ORCs are generated by voltage-gated Na- currents and T-type Ca2- currents in the somatic membrane. Isolated ORCs that have lost their cilia during the dissociation procedure are known to exhibit spike frequency accommodation by injecting the steady current. This raises the possibility that somatic ionic channels in ORCs may serve for odor adaptation at the level of spike encoding, although odor adaptation is mainly accomplished by the ciliary transduction machinery. This review discusses current knowledge concerning the mechanisms of spike generation in ORCs. It also reviews how neurotransmitters and hormones modulate ionic currents and action potentials in ORCs.

In the failing heart, numerous changes occur in cardiac adrenergic receptors (ARs) and intracellular signal transduction pathways. The most striking of these alterations appears at beta1 ARs, and the desensitization is the most prominent. Since malfunctions of beta1 ARs prevent intracellular signal transduction, the desensitization plays an important role in the onset and progression of the heart failure. Currently, several lines of evidence show the efficacy of inotropic agents, such as adenylate cyclase activator, that depend not on the ARs. Thus, it is essential to understand the pathway for the etiologic/pathologic evaluation for appropriate usage of these drugs for an adequate period. A novel water-soluble forskolin derivative, colforsin daropate hydrochloride (CDH) is a positive inotropic agent for treatment of the heart failure, especially in the severe stage with the beta1 AR desensitization. CDH potentiates cAMP activity via its direct action on adenylate cyclase, resulting in cardiotonic action. On the other hand, CDH relaxes vascular smooth muscle, while it antagonizes antidiuretic effects of angiotensin II and noradrenaline, involved in renal protection. In addition, CDH attenuates the mesangial cell proliferation and the inflammatory reaction, related with antiproliferative property of adrenomedullin and ketamine. To gain insights into the CDH action, we should take into account that intracellular signal transduction pathways in myocardium, smooth muscle and mesangial cell are controlled in a distinct manner.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic bone formation in the spinal ligaments. It has been suggested some role of mechanical stress in the progression of OPLL. Differential display RT-PCR was carried out to identify the genes participating in OPLL. A cDNA fragment corresponding to PGI2 synthase was highly expressed in OPLL cells compared to non-OPLL cells. To examine the effect of mechanical stress on the PGI2 synthase expression, cells were subjected to cyclic stretch and PGI2 synthase expression was assessed by quantitative RT-PCR. Cyclic stretch induced a time-dependent increase in PGI2 synthase in OPLL cells but not in non-OPLL cells. The increase in PGI2 synthase was diminished by SQ22536, a potent adenylate cyclase inhibitor. Cyclic stretch also induced PGI2 production. Beraprost and dibutyryl cAMP increased the mRNA expression of alkaline phosphatase (ALP) as a marker for osteogenic differentiation in OPLL cells, whereas no change was observed in non-OPLL cells. Beraprost- and stretch-induced increases in ALP expressions were inhibited by SQ22536. These data suggest that PGI2 synthase activated by mechanical stress plays a key role in the progression of OPLL, at least in part through the osteogenic differentiation in spinal ligament cells via the PGI2/cAMP system.

The effects of vanadate on lipoprotein lipase (LPL), a lipid-metabolizing enzyme, were tested using isolated rat fat pads. Vanadate increased the cellular LPL content through the stimulation of intracellular transport of the enzyme for activation, probably glycosylation. The stimulated release of LPL from the fat pads by vanadate was due to the increase in intracellular Ca2+ concentration, leading to the fusion of plasma membrane with vehicle included active LPL. Although vanadate shows insulin- and heparin-mimicking effects, it appears to differ from both insulin and heparin with regard to the mechanism of action. In isolated mouse fat pads, vanadate decreased the cellular leptin content and secretion by the increased degradation via a cAMP/PKA-dependent process involving proteasome activation and/or ubiquitination. This was the reverse of the action of insulin. In hepatocytes, cAMP phosphodiesterase type 3 activity was stimulated via the increased mitogen-activated protein kinase activity by vanadate. On the other hand, the stimulation by insulin was dependent on Akt kinase activation. The effects of vanadate were additive to those of insulin, suggesting that vanadate differs from insulin with regard to the receptor-signaling cascade. Furthermore, vanadate showed antiplatelet and antithrombin activity, leading to the prolongation of blood clotting time.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.

Aeromonas sobria has been recognized as pathogens associated with acute gastroenteritis in both adults and children. The major virulence factor has been proposed to be hemolysin which possesses both hemolytic and enterotoxic activities. Mature (bio-active) hemolysin secreted out of cells binds to the target cells of the host and injure the cells. However, hemolysin remained in bacteria can not express such toxicity. It means that the maturation and secretion pathway of hemolysin is closely related to the pathogenicity of bacteria. Therefore, I examined the pathway and clarified the following events. Hemolysin synthesized in cytoplasm translocates across the inner membrane and appears in a periplasmic space. Hemolysins appeared in the space associates to form dimer in the space. The C-terminal region of hemolysin functions as a trigger in the association. Dimerized hemolysin crosses the outer membrane and emerges in milieu, but monomer can not cross it. Therefore, the C-terminal region of hemolysin attributes not only to the formation of the dimer but also to its secretion into milieu. Hemolysin emerged in milieu is inactive. Inactive hemolysin is converted to bio-Active hemolysin by deleting its carboxyl-terminal 42-amino-acid peptide. Active hemolysin generated binds to the receptor of the target cell and stimulates the production of cyclic AMP by the cell. I assume that this stimulation closely relates to the induction of diarrhea by hemolysin.

Airway inflammation leads to secretion of abnormal mucous glycoprotein and ciliary injury. To investigate the possible usefulness of carbocisteine against airway inflammation and events related to it, we conducted a study in SO2-exposed rats of the effects of carbocisteine and ambroxol, as an active control drug, on components of mucous glycoprotein (fucose, sialic acid and protein) in bronchoalveolar lavage fluid (BALF); on infiltration and activation of inflammatory cells in BALF; on tracheal and bronchial-ciliary lesions; and on cAMP levels in tracheal and alveolar tissues. Carbocisteine inhibited or improved all SO2-induced changes tested, and dosages of 125 and 250 mg/kg b.i.d. reduced fucose, sialic acid and protein contents, inflammatory cells (as markers of inflammation), free radicals, and elastase activity in BALF, and suppressed the development of ciliary lesions of the tracheal and bronchial mucosa, while ambroxol (10 mg/kg b.i.d.) showed no such effects. In addition, carbocisteine improved cAMP levels in the tracheal and alveolar tissues. These results indicate that carbocisteine is able to prevent the development of inflammation-related respiratory disease in this rat model, and that this remission of airway inflammation may be associated with carbocisteine-induced normalization of cAMP levels in tracheal and alveolar tissues as well as with its mucoregulant and anti-inflammatory effects. In conclusion, carbocisteine has a unique mucoregulant action and inhibits SO2-induced airway inflammation in a manner different from that of ambroxol.

The kidney contributes to cardiovascular homeostasis through Na+ and water excretion and renin secretion. Changes in renal functions, therefore, have a close relationship to pathophysiology of cardiovascular diseases and to drug efficacy for them. The functions of the kidney are controlled by the sympathetic nervous system and various kinds of humoral factors and by their complicated interactions. Studies in the intact and working kidney in vivo have been providing physiologically significant information on the renal functions and drug actions. This review, by demonstrating data obtained in our laboratory with experiments in anesthetized dogs in vivo, refers mainly to the neural control of renal functions and renal actions of atrial natriuretic peptide (ANP) and an adenylate cyclase activator NKH477, either of which could be used for the treatment of congestive heart failure. Electrical stimulation of the renal nerves, which could mimic the events during elevation of renal sympathetic nerve activity, induces frequency-dependent renal norepinephrine release, renal vasoconstriction, antinatriuresis and renin secretion. ANP causes potent natriuresis and suppresses the nerve stimulation-induced renin secretion and renal vasoconstriction without affecting the norepinephrine release. Effects of ANP on other vasoconstrictive and antinatriuretic stimuli such as angiotensin II and endothelin are also demonstrated. Renal actions of NKH477 had been unknown, but we revealed that NKH477 elevates renal cAMP level and causes vasodilation and natriuresis. NKH477 also suppresses the nerve stimulation-induced renal vasoconstriction, and thereby blunts the antinatriuresis. The renal actions of these drugs clarified in our study may contribute to their curative effects on congestive heart failure.

The kidney plays a pivotal role in cardiovascular homeostasis through control of extracellular fluid volume, the mechanisms of which have been studied in detail at both cellular and molecular levels in each portion of the kidney. Renal clearance experiments in vivo are useful for analyzing renal functions as the integrated system of vascular, glomerular and tubular components. Physiological responses and drug effects within the kidney can be more clearly analyzed by measuring renal release of endogenous substances and by applying intrarenal arterial drug administration. For example, our recent studies in anesthetized dogs revealed the participation of phosphodiesterase IV in regulation of renal cAMP level and the influences by changes in renal cAMP level on renal hemodynamics, glomerular filtration and tubular reabsorption. The in vivo experiments also allow us to evaluate a sequence of events in the neural control of renal functions. We observed that an adenylate cyclase activator counteracted hypofiltration to attenuate antinatriuresis during sympathetic activation without inhibiting neurotransmitter release. The experiments in the in vivo kidney, although they provide only indirect information on intracellular events, are necessary for understanding the total renal functions and could thereby contribute to physiology and pharmacology of the cardiovascular system.

Adrenomedullin(AM) is a potent hypotensive peptide originally isolated from human pheochromocytoma. AM exerts various biological actions such as vasodilation, bronchodilation and natriuresis, by stimulating cAMP production and increasing free Ca2+ levels through the specific receptors. Although an orphan receptor cloned from rat lung, which contained seven transmembrane domains, was proved to be one of the AM receptors, it is now considered by many studies that other receptor subtypes should be present. The precise signal transduction mechanism for the AM receptor is not fully elucidated yet, but it is supposed that AM acts against proliferative changes of vascular and mesangial cells as seen in hypertensive states, at least partly by inhibiting the MAP kinase pathway. Further studies on the AM receptor subtypes and their intracellular signaling mechanisms are needed to clarify the role of AM in various pathophysiological conditions.

Familial male-limited precocious puberty(FMPP) is an autosomal dominant disorder characterized by marked elevation of serum testosterone despite low levels of gonadotropin. In 1993, a single point mutation, Asp578 to Gly(D578G), in the luteinizing hormone(LH) receptor gene was found in FMPP families. After discovery of the D578G mutation in the sixth transmembrane region of the LH receptor gene, seven other mutations in the fifth and sixth transmembrane regions, two mutations in the third intracellular loop and one mutation in the second transmembrane domain of the LH receptor have been found in the patients with familial and sporadic male-limited precocious puberty. These mutations caused constitutively elevated cAMP levels in transfected cells in vitro. These results suggested that Leydig cell activation and precocious puberty were caused by activating mutations of the LH receptor.

To examine possible contributions of beta(3)-adrenoceptors to catecholamine-induced pulmonary vasodilation, we studied isolated canine pulmonary arterial segments under isometric conditions. Addition of beta-adrenoceptor agonists produced a concentration-dependent relaxation of tissues precontracted with 50 mM KCl; the rank order of potency was isoproterenol (ISO, 1) > salbutamol (SAL, 0.97) > selective beta(3)-adrenoceptor agonists CL 316243 (CL, 0.87) and BRL 37344 (BRL, 0.86). Relaxant responses to SAL were competitively antagonized by the beta(2)-adrenoceptor antagonist ICI 118551 and the pA2 value was 6.67 +/- 0.21 (mean +/- SE), whereas the response to CL was weekly antagonized only by a high concentration of ICI 118551 (10(-5) M) and the apparent pA2 value was 5.24 when alpha-and beta(1)-adrenergic receptors were blocked. By contrast, the atypical beta-adrenoceptor antagonist cyanopindolol antagonized CL-induced relaxation in a competitive manner; the pA2 value was 6.71 +/- 0.12, which was lower than that with salbutamol (p < 0.05). Intracellular cyclic AMP levels were increased in a contraction-dependent manner by CL. These results suggest that beta(3)-adrenoceptors may exist in canine pulmonary arterial smooth muscle and that stimulation of this atypical receptor causes vasodilation through a cyclic AMP-dependent pathway.

The DNA of AVP vasopressin (AVP) is composed of 3 exons (A, B, C) and 2 introns (I, II). Following transcription, translation, and processing AVP(MW 1081) is produced. AVP receptors (VR) are classified into V1aR, V1bR and V2R. V1aR is composed of 418 amino acids (AA) and its MW is 46745. V1aR is in the vascular smooth muscle (VSM) and activates the phosphatidylinositol system, leading to the contraction of VSM. V1bR is composed of 424 AA and its MW 47034. It's second messenger systems are obscure, but V1bR is responsible for ACTH release. V2R is composed of 371 AA and its MW 40285. V2R is in the kidney and enhances the production of cAMP, leading to renal water and urea reabsorption.

To determine the effect of time in culture on epithelial cell function, we evaluated the modulation of Na(+)-K(+)-ATPase activity in rat alveolar type II cells in culture. Ouabain sensitivity testing revealed that the alpha-1 predominance in the enzyme's isoforms was maintained over the 120 hours in culture. Basal Na(+)-K(+)-ATPase activity in the whole cell homogenate did not differ significantly between cells cultured for 48 hours and those cultured for 120 hours. Terbutaline (10 mM) did not activate Na(+)-K(+)-ATPase in the cells cultured for 48 hours, but, it significantly increased the activity of this enzyme in the cells cultured for 120 hours cells cultured for 48 hours, produced intracellular cyclic AMP after exposure to 10 mM of terbutaline. These results indicate that the coupling between Na(+)-K(+)-ATPase and the beta-adrenergic pathway in alveolar type II cells can be influenced by the time in cell culture.

The elucidation for the mechanism of receptor-mediated signal transduction has been the aim of our extensive studies. Cyclic AMP, which was controlled by membrane adenylyl cyclase, was an intracellular signal (the first second messenger in cells proposed by Sutherland) given by hormones and neurotransmitters. The GTP-binding (G) proteins play an important role in communication between membrane receptors and the adenylyl cyclase catalytic unit. One (Gs) of the G proteins is involved in the activation, while the other (Gi) is involved in the inhibition of adenylyl cyclase. Islet-activating protein (IAP, pertussis toxin, PTX) catalyses the transfer of the ADP-ribose moiety of NAD to the alpha subunit of Gi, resulting in a complete loss of the Gi functions. In some cases, arachidonic acid (AA) regulates cell functions as a second messenger. AA serves as a precursor to a number of biologically active lipids including prostaglandins and leukotrienes. Activation of cell surface receptors of many cell types results in the release of AA from membrane phospholipids by phospholipase A2 (PLA2). A new family of PLA2 has been discovered in the cytosol of various cells. The activation of receptor-mediated AA release by cytosolic PLA2 was also regulated by PTX-sensitive G proteins. PTX treatment inhibited cell growth of fibroblasts by serum and growth factors. G proteins have been involved in receptor-receptor interactions in neuronal cells. These findings suggest the regulatory roles of cell surface receptors-coupled G proteins in signal transductions and cell functions.

In gastric parietal cells the histamine H2 receptor is involved in gastric acid secretion. Molecular cloning of the cDNA for the histamine H2 receptor has revealed that the receptor is a member of the G-protein coupled receptor family. We expressed histamine H2 receptors in CHO cells, COS7 cells, and Sf9 cells to analyze N-glycosylation, palmitoylation, and desensitization of the H2 receptor, which are difficult to analyze in isolated parietal cells. The findings obtained in these systems will be useful for studying the gastric acid secretion mechanisms in parietal cells.

Transcription of a number of eukaryotic genes is activated in response to an increase in the intracellular cAMP concentration. These genes stimulated by cAMP have a common promoter element, cAMP response element (CRE). The CRE is recognized by a CRE binding protein, CREB. The binding of CREB to CRE does not induce transcription. Activation of transcription requires the phosphorylation or CREB at Ser-133. In the case of the cAMP pathway, the activated catalytic subunit of cAMP-dependent protein kinase (PKA) translocates to the nucleus and phosphorylates Ser-133 of CREB. In the nervous system, signals transmitted across synapses are known to regulate gene expression in the post-synaptic cell. This process often involves membrane depolarization and subsequent amplification of intracellular Ca2+. The transcriptional activation induced by membrane depolarization and Ca2+ influx is mediated by a promoter element, called the Ca(2+)-responsive element (CaRE). Recent studies of c-fos and proenkephalin gene expression have shown that the CaRE is indistinguishable from a CRE. In this paper, we focus on the possible interactions between Ca2+ and the cAMP signaling pathways into the nucleus.

Xenopus oocytes has been utilized to analyze the intracellular signaling and coupling mechanisms between neurotransmitter receptors and ion channels. (1) The GTP-binding protein-coupled intracellular signaling pathway was analyzed in oocytes expressing metabotropic receptors by brain mRNA. These metabotropic receptors are commonly linked to the sequence of phosphoinositide metabolism, intracellular Ca2+ increase and opening of Ca(2+)-dependent Cl- channels. An antisense DNA study indicated that a specific subtype of GTP-binding protein is involved in the coupling of each metabotropic receptor. (2) Effects of central acting drugs on the functions of glutamate receptor subtypes and voltage-dependent Ca2+ channels were evaluated, and the data were compared with the results from conventional in vitro assays using brain preparations. (3) A series of experiments on kappa-opioid receptors indicated that kappa-opioid receptors can couple with multiple signaling systems in the oocytes via GTP-binding proteins Gi/G(o), which involves mobilization of intracellular Ca2+ through phosphoinositide metabolism, synergistic potentiation of cyclic AMP production, and inhibition of voltage-dependent Ca2+ channels.

Synaptoneurosomes are entities in which a presynaptic sac (synaptosome) is attached to a resealed postsynaptic vesicle (neurosome). Biochemical studies have demonstrated that the synaptoneurosomes retain several receptor-mediated activities such as accumulations of cyclic AMP and ion fluxes. GABAA receptor-mediated 36Cl- uptake and modulatory effects of the uptake by benzodiazepines, barbiturates and neurosteroids have been reported in rat brain synaptoneurosomes. The in vitro protocol of the GABAA receptor ligand-stimulated 36Cl- influx proves to be of value for the screening of new compounds such as anxiolytics.

The secretion of parathyroid hormone (PTH) is influenced by various factors, but extracellular calcium concentration ([Ca2+]e) is the sole factor established to be the physiological regulator. [Ca2+]e acts on Ca2+ sensing receptor recently cloned, and elevates intracellular calcium ([Ca2+]i) and inositol trisphosphate. Although the changes in cAMP level and C-kinase activity also have been demonstrated, the mediator directly operating secretory mechanism of PTH has not been defined. Recently, the activity of chromogranin A-related peptides to modulate PTH secretion was found and their role as the autocrine-paracrine regulator of PTH secretion has been proposed.

Various hormones and neurotransmitters as well as glucose are known to increase the cAMP concentration in pancreatic beta cells. To determine the mechanism by which cAMP augments insulin gene expression, we first identified the cAMP response elements (CREs) of the human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, we constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyltranferase (CAT) gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma cells (HIT) showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter.

The thyroid stimulating antibodies (TSAb) have been widely used as a clinical marker for diagnosis of hyperthyroidism due to Graves' disease. Immunoglobulins (IgG) of Graves' patients as well as TSH is known to stimulate cAMP production in FRTL-5 cells, a cell-line derived from normal rat thyroid. On the other hand, it has been shown that TSH-induced I- efflux in these cells is associated with production of inositolphosphate as well as intracellular Ca2+ increase. An attempt was made to determine whether Graves' IgG also can mimic such TSH action as a cause of Graves' disease. Polyethyleneglycol precipitated serum fraction of Graves' patients were prepared. The cells grown in the presence of TSH were incubated for 3 weeks in a Ham's 10 medium containing 1% calf serum, insulin and hydrocortisone for TSH depletion. After preincubation with 125I-iodide for 50 min to label intracellular iodide, the cells were challenged by serum samples for 1 min. The addition of normal pooled serum hardly affected the I- efflux. The Graves' immunoglobulin G fractions stimulated I- efflux dose-dependently. The mean potency of 12 patient's sera relative to the values of the pooled serum as 100% was 217 +/- 56.4%, which was significantly higher (p < 0.001) than that for 8 normal subjects (110 +/- 16.1%). The results obtained herein indicate that IgG of Graves' patients activate both adenylate aclase-cAMP system and a phospholipid-Ca2 system. I- efflux stimulating immunoglobulins (IESI) activity is a useful marker in making a diagnosis or determining a remission of hyperthyroidism due to Graves' disease.