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Free magnesium ion (Mg(2 + )) is involved in numerous processes of cardiac function. However, mechanism of regulation by Mg(2 + ) has not been fully understood. Extracellular Mg(2 + ) can act on the external surface of the cell membrane, whereas intracellular Mg(2 + ) can exert its effects via many different sites : various enzymes, intracellular organella and internal surface of the cell membrane. In this article, we will briefly review the extracellular and intracellular effects of Mg(2 + ) on each step of E-C coupling of cardiac myocytes, in an attempt to integrate them into cardiac function.

Endothelial cells lining blood vessels are in tight contact with each other, thereby maintaining vascular integrity. Compromising vascular integrity leads to an increase in vascular permeability, which is associated with chronic inflammation, edema, and tumor angiogenesis. Vascular endothelial (VE)-cadherin is an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier functions. We previously reported that cyclic AMP-elevating agonists such as prostaglandins and adrenomedullin potentiate VE-cadherin-dependent cell adhesion by inducing activation of Rap1 small GTPase through Epac. We further investigated the mechanism whereby Rap1 potentiates VE-cadherin-dependent cell adhesion, and found that Rap1 induces the formation of circumferential actin bundles along the cell-cell junctions. Although it has been believed that α-/β-catenins anchor cadherin to the actin cytoskeleton to stabilize cadherin at cell-cell junctions (classical model), Nelson's and Weis' groups have recently suggested a new dynamic model in which α-/β-catenins do not stably connect actin to cadherin. However, our study clearly indicated that the circumferential actin bundles anchor VE-cadherin to the cell-cell junctions through α-/β-catenins. Thus Rap1 potentiates endothelial cell-cell junctions through the mechanism based on the static model.

Vascular tone is tightly regulated by endothelium-derived factors. These include relaxing factors (EDRFs) such as nitric oxide (NO), hyperpolarizing factors (EDHFs), and contracting factors (EDCFs). Although EDHF is a prominent vasodilator, particularly in smaller arteries, little attention has been paid to the potential role of EDHF responses in diabetes. EDHF signaling may involve various factors, including several diffusible factors and non-diffusible factors (e.g., gap junctions). It has been demonstrated that the alterations in EDHF relaxation seen in mesenteric arteries from diabetic rats may be attributable to an increase in phosphodiesterase 3 (PDE3) activity, leading to a reduction in the action of adenosine 3',5'-cyclic monophosphate (cAMP), and consequently the activity of protein kinase A (PKA) is decreased in such arteries. Moreover, it has been suggested that the imbalance between EDRFs and EDCFs is present in mesenteric arteries from type 2 diabetic rats and the EDHF relaxation can be partly reversed by suppression of EDCF signaling. Indeed, chronic treatment with metformin, eicosapentaenoic acid, or thromboxane synthase inhibitor can reduce EDCF signaling and normalize EDHF signaling in mesenteric arteries from type 2 diabetic rats. Although the improvement or restoration of EDHF responses has not been the direct subject of any pharmaceutical effort, increasing cAMP/PKA signaling (e.g., by inhibiting PDE3 activity) or reducing EDCFs signaling has potential as an interesting therapeutic target in diabetic vasculopathy.

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.

Olfactory transduction is achieved by the G-protein-mediated molecular system. For a long period of time, research on the olfactory transduction system has been limited to qualitative descriptions, because of experimental difficulties accompanying use of diverse ligands, multiple receptors and the fine structure of the sensory cilia (200 nm diameter). However, recent works gradually overcame those difficulties to reveal the molecular systems within nano-scale biological structure. When the cAMP dynamics were estimated, the cAMP-production rate was on the order of 20,000/s/cilium at the maximum odorant stimulus. This number is much smaller than that known for other systems, and therefore it is suggested that the olfactory enzymes use low signal amplification. Apparently, signal transduction with a small number of molecules is achieved by the fine ciliary structure that has a high surface-volume ratio in which even a small change in the absolute number of molecules is reflected in a big change in concentration. In addition, the olfactory receptor cell has a unique and strong non-linear amplification detecting a slight change in the odorant dose, which is regulated by Ca2- that flows through the CNG channel; a cytoplasmic increase of Ca2- in turn activates excitatory Cl current to boost the net transduction current.

Neurotrophic factor-like substances and inducers of neurotrophic factor biosynthesis have enormous therapeutic potential for serious neuronal diseases such as Alzheimer's disease. Here, we discuss about the pharmacological effects of scabronines and beta-eudesmol as promising candidates of leading compounds against the dementia. In addition, we discuss about the signaling pathway of nerve growth factor and cyclic AMP in order to seek the signaling molecules as targets of drug development.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a ubiquitous neuropeptide in the central and peripheral nervous systems. Previously we reported that PACAP38 is localized in pancreatic islets and serves as an endogenous amplifier of glucose-induced insulin secretion. PACAP activates Gs-cAMP system, stimulates voltage-dependent Ca(2+) channels, and increases cytosolic Ca(2+) concentration in beta-cells. On the other hand, PAC1 receptor is expressed in adipocytes. PACAP enhances insulin-stimulated glucose uptake in an adipocyte cell-line, 3T3-L1 cells. PACAP does not alter the tyrosine phosphorylation of insulin receptor and IRS-1, but increases the activity of PI-3 kinase, a distal site of insulin signaling. PACAP also promotes differentiation of 3T3-L1 cells from fibroblasts to adipocytes. In GK rats, an animal model of type 2 diabetes, daily i.p. injection of PACAP38 (6 pmol/kg) from the age of 3 weeks prevents development of hyperglycemia between 3 to 8 weeks. These results demonstrate that PACAP enhances glucose-stimulated insulin secretion in islets, enhances insulin action inadipocytes, and prevents hyperglycemia in diabetic animals. This finding presents a possible therapeutic use of PACAP in the treatment of diabetes.

Advances in the neurosciences over the past two decades have elucidated that alcoholism is a chronic and easily recurring disorder, which is based on brain damage induced by long-term ethanol consumption. Researchers have identified neural circuits that subsume the actions of ethanol and they have also elaborated many of the intracellular signalling pathways that follow receptor activation by ethanol, suggesting a substantial difference between the addicted brain and non-addicted brain. However, most of the molecular biological changes, which are likely based on the transition from occasional drinking to alcohol dependence, remain to be elucidated. This review summarizes the findings on the biological background of alcoholism concerning the brain rewarding system, its related receptors, and the second messenger-mediated signal transduction system within the cells, especially focusing on the cyclic AMP signaling pathway calcium (Ca2+) system in neurons. In addition, our studies with neural stem cells are introduced to indicate future directions of research on the pathophysiology of alcoholism.

MaxiK channel, the large conductance Ca(2+)-sensitive K+ channel, is expressed abundantly in vascular smooth muscles and plays a key role in the tuning of their excitability and contractility. The present study was carried out to elucidate the contribution of MaxiK channel to prostacyclin receptor (IP receptor)-mediated vascular relaxation with a special reference to the role of cAMP. An IP agonist, beraprost, induced a strong relaxation in de-endothelialized guinea pig thoracic aorta, which was almost abolished by a MaxiK channel selective blocker, iberiotoxin (IbTx). Beraprost produced a 30-fold rise in tissue cAMP contents. In addition, beraprost-induced relaxation potentiated in the presence of Ro-20-1724 (a selective inhibitor of cAMP-specific phosphodiesterase) was completely counteracted by IbTx. However, beraprost-induced relaxation was not affected by SQ22,536, an adenylyl cyclase inhibitor, which abolished this IP agonist-induced elevation of cAMP contents. SQ22,536-insensitive relaxant component was significantly inhibited by IbTx. Cholera toxin, a Gs activator, qualitatively mimicked the effects of beraprost. Furthermore, MaxiK channel currents in aortic myocytes were increased by beraprost in a GTP-dependent manner. These results indicate that both cAMP-dependent and -independent pathways contribute to MaxiK channel-mediated vascular relaxation following IP receptor stimulation. Direct regulation by Gs seems to partly account for MaxiK channel-mediated, cAMP-independent mechanism.

Four prostaglandin E (EP) receptor subtypes have been identified and cloned, designated as EP1, EP2, EP3 and EP4. These EP receptors are members of the G-protein coupled receptor family. EP3 receptor signals are primarily involved in inhibition of adenylyl cyclase via Gi activation, while EP2 and EP4 receptor signals cause a stimulation of adenylyl cyclase via Gs activation. Immune cells, such as mast cells, express multiple EP subtypes on their cell membranes, but few studies have been conducted to understand exactly what signals the main flow for the multiple subtypes expressing immune cells. We previously demonstrated that activation of Gi-coupled EP3 receptor exhibited a cooperative effect on cAMP synthesis induced by Gs-coupled EP2 receptor in COS-7 cells. Here we report that a selective EP4 agonist-induced adenylyl cyclase activity was augmented by simultaneous addition of a selective EP3 agonist in mastocytoma P-815 cells, which express mRNAs for both EP3 and EP4 subtypes. The augmentation in cAMP synthesis was found to be pertussis toxin-sensitive. P-815 cells are demonstrated to bind to Pronectin-F, a proteolytic fragment of fibronectin, in adhesion protein of the extracellular matrix, by addition of PGE2, which is mediated by PKA. The binding of P-815 cells to Pronectin-F mediated by EP4 receptor was augmented by the EP3 receptor. These findings indicate that two subtypes of PGE2 receptors, EP3 and EP4, cooperatively activate the cAMP-mediated adhesion event through induction of fibronectin ligand elicited by PGE2 in P-815 cells. Furthermore, the PGE2-induced adhesion response may contribute to the mast cell recruitment function on extracellular matrix during inflammation.

This article describes current information concerning analyses of contraction and relaxation associated with electrical stimulation of efferent nerves in isolated mammalian sphincter muscles. Contractile responses of sphincters are mediated by alpha 1-adrenoceptors and muscarinic receptors stimulated by transmitters from adrenergic and cholinergic nerves, respectively, whereas those of the bladder body are almost exclusively mediated by transmitters from parasympathetic nerves. Relaxant responses to nerve stimulation are ascribed mainly to mechanisms that are sensitive and resistant to nitric oxide (NO) synthase inhibitors. Neurogenic calcitonin gene-related peptide and beta-adrenoceptor activation by neurogenic norepinephrine may also be involved in some mammals. Stimulus frequency is an important determinant to distinguish NO synthase-sensitive and -resistant components; responses at low frequencies are abolished by the enzyme inhibitors, whereas those at high frequencies are inhibited only partially. High and low frequency stimulation increases the cyclic GMP content in muscles, suggesting the involvement of neurogenic NO, although relaxation at high frequencies is only partially due to such a mechanism. From pharmacological studies so far analyzed, including ours performed with porcine urinary tract sphincters, it is concluded that NO synthase resistant-relaxation is not mediated by peptides nor compounds that open K+ channels in muscle cell membrane and stimulate beta-adrenoceptors. Contribution of NO and non-NO relaxing factor (s) in relaxant responses varies with animal species. Identification of this factor, determination of intracellular signaling processes and interaction with the NO/cyclic GMP system may give us a clue in developing new therapeutics to treat dysfunctions of the lower urinary tract sphincters.

Tetrahydrobiopterin(BH4) serves as an essential cofactor for the biosynthesis of nitric oxide (NO). BH4 is de novo synthesized from GTP and GTP cyclohydrolase I(GCH I) is the rate-limiting enzyme in the biosynthesis of BH4. Under inflammatory conditions, it is reported that endothelial cells release large amount of BH4. In this study, we examined the regulation mechanism of the biosynthesis of BH4 in human umbilical vein endothelial cells(HUVEC). Prostacyclin and forskolin, reagents of stimulation of cAMP signaling cascade, reduced cytokine induced biosynthesis of BH4 through the inhibition of expression of GCH I mRNA. On the other hand, stimulations of NO-cGMP signaling pathway inhibited GCH I activities through the post translational modification of GCH I enzyme. Both two signaling cascade lead to vasodilation. It is suggested that the biosynthesis of BH4 can be regulated by negative feed back regulation systems between endothelium and smooth muscle cells to prevent over stimulated vasodilation.

beta-Adrenoceptor subtypes which mediate relaxation of guinea-pig gastrointestinal smooth muscles in response to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and beta 3-adrenoceptor agonists (BRL37344 and (+/-)-CGP12177A) are predominantly beta 3-adrenoceptors. Although cAMP-PKA system is thought to play a substantial role in the smooth muscle relaxation mediated via beta 1- and beta 2-adrenoceptors, there is little information on the role of cAMP in beta 3-adrenoceptor-meidated relaxation. The present study was carried out to elucidate the role of cAMP in beta 3-adrenoceptor-meidated relaxation of guinea-pig gastric fundus smooth muscle. Furthermore, possible contribution of two types of K+ (voltage-dependent and Ca(2+)-activated K+, BKCa; voltage-dependent, Kv) channels was also examined pharmaco-mechanically. In gastric fundus smooth muscle, catecholamines and beta 3-adrenoceptor agonists elicited potent relaxations in the presence of beta 1- and beta 2-adrenoceptor antagonists. All of these relaxations were not diminished by an adenylate cyclase inhibitor, SQ-22,536 (100 microM), which indicates their characteristic of cAMP-independency. SQ-22,536-resistant, beta 3-adrenoceptor-mediated relaxations were strongly attenuated by a Kv channel blocker, 4-aminopyridine (3 mM), but not by iberiotoxin (100 nM), a selective blocker of BKCa channel. The present results indicate that 4-aminopyridine-sensitive Kv channels play a primary role in cAMP-independent relaxation of guinea-pig gastric fundus smooth muscle in response to the stimulations of beta 3-adrenoceptors.

We investigated the possible mechanisms of development of latent learning and morphine dependence by the methods of behavioral pharmacology and confirmed them by using mutant mice. The heterozygous mice for the tyrosine hydroxylase (TH) gene and for the cyclic AMP (cAMP) response element binding protein (CREB) binding protein (CBP) gene showed the impairment of latent learning in the water finding task, and these mice did not develop morphine dependence. The spatial learning and hippocampal long-term potentiation (LTP) were normal in the both mutants. TH heterozygous mice showed a reduction of high K(+)-evoked noradrenaline release in the frontal cortex measured by the microdialysis technique and of cAMP content in the brain. In conclusion, the results of mutant mice suggest that the alternation of catecholamine biosynthesis and cAMP signal pathways may play a key role in development of latent learning and morphine dependence, and they furthermore show that the expression of genes mediated by phosphorylated CREB may be involved in the development of latent learning and morphine dependence.

Studies on the cellular and molecular mechanism of neurotransmitter receptor-signaling and of neuronal and glial cell responses to stresses seem to be important to elucidate the action mechanism of centrally-acting drugs and to develop novel therapeutics against several diseases in the brain. The present review shows our findings with regard to the membrane receptor-signaling mechanism including serotonin, noradrenaline, glutamate receptors, ion channels, G-proteins, protein kinases and drug actions in Xenopus oocytes injected with rat brain mRNA, NG108-15 cells and brain membranes. Regarding the results of studies on the inter- and intra-cellular mechanism of neurons and glial cells against cerebral ischemia/hypoxia, we review the involvement of a transcription factor NF-kappa B in LPS-elicited inducible NO synthase (iNOS) expression in rat astroglial cells. Then we describe possible involvement of: 1) ADP-ribosylation/nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 2) decrease in mitochondrial membrane potential, release of caspase-3 from mitochondria and degradation of the inhibitor of caspase-activated DNase by activated caspase in NO-induced neuronal apoptosis. We observed that hypoxia results in expression of a molecular chaperon such as protein disulfide isomerase (PDI) and HSP70 in astroglial cells. Our recent findings indicate that overexpression of PDI in the rat hippocampus (in vivo) and in neuroblastoma SK-N-MC cells (in vitro) significantly suppress the hypoxia-induced neuronal death. From physiological/pathophysiological and pharmacological aspects, we review the importance of studies on the cellular and molecular mechanism of membrane receptor-signaling and of stress-responses in the brain to identify functional roles of neuro-glial- as well as neuro-neuronal interaction in the brain.

Prostaglandin E2 (PGE2) acts as a potent stimulator of bone resorption. We examined PGE2-induced bone resorption using mice lacking each subtype (EP1, EP2, EP3 and EP4) of PGE receptor and identified the PGE receptor subtype(s) mediating PGE2 action. In calvarial culture from EP1-, EP2-, and EP3- knockout mice, PGE2 stimulated bone resorption to a similar extent to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption in response to PGE2 was found in the calvarial culture from EP4-knockout/mice. DbcAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. In mouse calvarial cultures, EP4-agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2-agonist also stimulated bone resorption, but only slightly, EP1- and EP3-agonists did not stimulate it at all. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP, which is mediated mainly by EP4 and partially by EP2.

To investigate the involvement of catecholamines and/or the cyclic AMP (cAMP) systems in the development of drug dependence, we examined whether morphine dependence was developed in tyrosine hydroxylase (TH) heterozygous (TH+/-) and cAMP response element binding protein (CREB) binding protein (CBP) heterozygous (CBP+/-) mice. Morphine (10 mg/kg) induced place preference in the wild-type mice. In the TH+/- and CBP+/- mice, however, we could not find any morphine-induced place preference. When the wild-type mice pretreated with morphine (10 mg/kg) twice a day for 5 days were challenged with naloxone (5 mg/kg), they showed increased numbers of jumping, rearing and forepaw tremor as a sign of withdrawal symptom and increased level of cAMP in the thalamus/hypothalamus, but not in the striatum. However, increased numbers of jumping and forepaw tremor in the TH+/- and CBP+/- mice and increased level of cAMP in the thalamus/hypothalamus of TH+/- mice were not observed. These results suggest that catecholamines and CBP are involved in the development of morphine dependence, and that some changes in the catecholaminergic and/or cAMP system induced by repeated morphine treatment play an important role in the addiction of morphine.

This paper will review 1) experimental models of drug-seeking behavior and 2) mechanisms underlying the behavior, focusing on cocaine self-administration. After the acquisition of self-administration, vigorous lever-pressing is generally observable after the drug was replaced by saline. This lever-pressing behavior under saline infusion can be considered "drug-seeking behavior". Drug-seeking behavior is reinstated by non-contingent injection of the drug, stress exposure and presentation of drug-associated stimuli even after extinction. This is called a relapse/reinstatement model. Electrophysiological studies showed that the majority of accumbal neurons is tonically inhibited during cocaine self-administration and exhibited phasic increases in firing time-locked to cocaine self-infusion, which might represent the craving state or drive animals to drug-seeking behavior. Voltammetry and microdialysis studies indicated that the timing of drug-seeking responses can be predicted from fluctuations in accumbal extracellular dopamine concentration. Whereas dopamine D2-like agonists reinstated extinguished cocaine-seeking behavior, D1-like agonists prevented the relapse in cocaine-seeking behavior induced by cocaine itself. Given that an AMPA receptor antagonist, but not dopamine antagonist, prevented cocaine-seeking behavior induced by cocaine, glutamate transmission in the nucleus accumbens is thought to be important for expression of craving or drug-seeking behavior.

cAMP and IP3 act as secondary messengers in olfactory signal transduction and when activated, stimulate calcium levels in olfactory receptor cells. Little is known however, about the causal mechanism. We studied calcium kinetics in mouse olfactory receptor cells after odorant stimuli. Olfactory receptor cells were isolated from female BALB/c mice, treted with trypsin, and stained with Fura-2/AM. Changes in intracellular Ca2+ concentrations in stained cells were measured with a fluorescent microscopic image-processing device (ARGUS-50; Hamamatsu Photonix, Japan). We found that intracellular Ca2+ concentrations rose after exposure to a set of odorants, including 3-ethoxy-4-hydroxy-benzaldehyde, caprylic acid, heptanoic acid, nonanoic acid, eugenol, phenethyl alcohol, and n-amyl acetate. Adding 2', 5'-dideoxyadenosine, a cAMP inhibitor, beforehand suppressed olfactory receptor cell response to odorants. Intracellular Ca2+ concentrations increased substantially in response to stimulation by odorants in calcium-free Ringer's solution, but only a slight increase was seen in intracellular calcium concentration in response stimulation by a high concentration of K+ (145.6 mM) in calcium-free Ringer's solution. The increase in intracellular Ca2+ concentration after odorant stimuli was suppressed when olfactory receptor cells were pretreated with ryanodine, which releases Ca2+ from intracellular stores. These findings suggest that elevated Ca2+ concentrations may be involved in releasing Ca2+ from intracellular calcium stores in mouse olfactory receptor cells, in which cAMP functions as a secondary messenger in olfactory signal transduction.

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.

Binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase (PKA) is an essential step for cAMP-mediated signal transduction including phosphorylation of cAMP response element binding protein (CREB). In the present study, binding activity of PKA with cAMP and CREB phosphorylation were examined in rat focal brain ischemia induced by occlusion of the middle cerebral artery for 1.5 hours followed by various time of recirculation. Binding activity of PKA with cAMP was progressively inhibited during the acute phase of ischemia from the ischemic core to peri-ischemia area. Phosphorylated CREB-positive cells in the ischemic core revealed a significant, but transient increase in number at 3.5 hours of recirculation, followed by a rapid decrease below the control level during the subsequent period. On the other hand, in the peri-ischemia area, the number of phosphorylated CREB-positive cells showed a more marked increase as compared to that in the ischemic core, and the increase continued until 48 hours of recirculation with a tendency for gradual decline. Persistent enhancement of CREB phosphorylation may thus be closely related to the neuronal viability and neuroprotective mechanisms, whereas rapid disappearance of CREB phosphorylation following ischemic insult may clearly precede neuronal death.