Ca²⁺ is a central player in the excitation-contraction coupling of cardiac myocytes, the process that enables the heart to contract and relax. Mishandling of Ca²⁺ is a central cause of both contractile dysfunction and arrhythmias in pathophysiological conditions such as heart failure (HF). Upon electrical excitation, Ca²⁺ enters the myocytes via voltage-gated Ca²⁺ channels and induces further Ca²⁺ release from the sarcoplasmic reticulum (SR). This raises the free intracellular Ca²⁺ concentration ([Ca²⁺]_i), activating contraction. Relaxation is driven by [Ca²⁺]_i decline, mainly due to re-uptake into the SR via SR Ca²⁺-ATPase and extrusion via the sarcolemmal Na⁺/Ca²⁺ exchange, NCX. Intracellular Na⁺ concentration ([Na⁺]_i) is a main regulator of NCX, and thus [Na⁺]_i plays an important role in controlling the cytosolic and SR [Ca²⁺]. [Na⁺]_i may have an even more important role in HF because NCX is generally upregulated. There are several pathways for Na⁺ entry into the cells, whereas the Na⁺/K⁺ pump (NKA) is the main Na⁺ extrusion pathway and therefore is essential in maintaining the transmembrane Na⁺ gradient. Phospholemman is an important regulator of NKA function (decreasing [Na⁺]_i affinity unless it is phosphorylated). Here we discuss the interplay between Ca²⁺ and Na⁺ in myocytes from normal and failing hearts.

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At present, nine adrenoceptor (AR) subtypes have been identified: α_1A-, α_1B-, α_1D-, α_2A-, α_2B-, α_2C-, β₁-, β₂-, and β₃AR. In the human heart, β₁- and β₂AR are the most powerful physiologic mechanism to acutely increase cardiac performance. Changes in βAR play an important role in chronic heart failure (CHF). Thus, due to increased sympathetic activity in CHF, βAR are chronically (over)stimulated, and that results in β₁AR desensitization and alterations of down-stream mechanisms. However, several questions remain open: What is the role of β₂AR in CHF? What is the role of increases in cardiac G_i-protein in CHF? Do increases in G-protein-coupled receptor kinase (GRK)s play a role in CHF? Does βAR-blocker treatment cause its beneficial effects in CHF, at least partly, by reducing GRK-activity? In this review these aspects of cardiac AR pharmacology in CHF are discussed. In addition, new insights into the functional importance of β₁- and β₂AR gene polymorphisms are discussed. At present it seems that for cardiovascular diseases, βAR polymorphisms do not play a role as disease-causing genes; however, they might be risk factors, might modify disease, and/or might influence progression of disease. Furthermore, βAR polymorphisms might influence drug responses. Thus, evidence has accumulated that a β₁AR polymorphism (the Arg389Gly β₁AR) may affect the response to βAR-blocker treatment.

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Sarcoplasmic reticulum (SR) Ca²⁺ cycling, that is, the Ca²⁺ clock, entrained by externally delivered action potentials has been a major focus in ventricular myocyte research for the past 5 decades. In contrast, the focus of pacemaker cell research has largely been limited to membrane-delimited pacemaker mechanisms (membrane clock) driven by ion channels, as the immediate cause for excitation. Recent robust experimental evidence, based on confocal cell imaging, and supported by numerical modeling suggests a novel concept: the normal rhythmic heart beat is governed by the tight integration of both intracellular Ca²⁺ and membrane clocks. In pacemaker cells the intracellular Ca²⁺ clock is manifested by spontaneous, rhythmic submembrane local Ca²⁺ releases from SR, which are tightly controlled by a high degree of basal and reserve PKA-dependent protein phosphorylation. The Ca²⁺ releases rhythmically activate Na⁺/Ca²⁺ exchange inward currents that ignite action potentials, whose shape and ion fluxes are tuned by the membrane clock which, in turn, sustains operation of the intracellular Ca²⁺ clock. The idea that spontaneous SR Ca²⁺ releases initiate and regulate normal automaticity provides the key that reunites pacemaker and ventricular cell research, thus evolving a general theory of the initiation and strength of the heartbeat.

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It is widely accepted that a high sodium intake triggers blood pressure rise. However, only one-third of the normotensive subjects were reported to show salt-sensitivity in their blood pressure. Many factors have been proposed as causes of salt-sensitive hypertension, but none of them provides a satisfactory explanation. We propose, on the basis of accumulated data, that the reduced activity of the kallikrein-kinin system in the kidney may provide this link. Renal kallikrein is secreted by the distal connecting tubular cells and all kallikrein-kinin system components are distributed along the collecting ducts in the distal nephron. Bradykinin generated is immediately destroyed by carboxypeptidase Y-like exopeptidase and neutral endopeptidase, both quite independent from the kininases in plasma, such as angiotensin converting enzyme. The salt-sensitivity of the blood pressure depends largely upon ethnicity and potassium intake. Interestingly, potassium and ATP-sensitive potassium (K_ATP) channel blockers accelerate renal kallikrein secretion and suppress blood pressure rises in animal hypertension models. Measurement of urinary kallikrein may become necessary in salt-sensitive normotensive and hypertensive subjects. Furthermore, pharmaceutical development of renal kallikrein releasers, such as K_ATP channel blockers, and renal kininase inhibitors, such as ebelactone B, may lead to the development of novel antihypertensive drugs.

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It had been believed that angiotensin II (Ang II) was produced by the renin-angiotensin system (RAS), which was established in the 1950’s. After a while, people realized that the multiple functions of Ang II could not be explained by the conventional RAS. We have tried to determine the existence of the tissue Ang II generating system. At first, we found that vascular angiotensin-converting enzyme (ACE) was increased to generate local Ang II in the vessels of hypertension and was enhanced in lipid-loaded atherosclerosis, to respond to ACE inhibitor or Ang II antagonist (ARB). In both cases, Ang II production in vessels was independent from the systemic RAS that was estimated by the plasma renin activity. On the way to clarifying the roles of the vascular ACE, we noticed that vascular Ang II production was not completely suppressed by ACE inhibitor alone. This evidence led us to discover different types of chymase as a new Ang II producing enzyme. Now, we have obtained a strategy to distinguish the Ang II one by one, that is, circulating RAS derived, tissue ACE derived, and chymase derived. It is essential to understand not only the intracellular mechanisms of Ang II but also the process of Ang II productions in each disease to show accurate indications of the effectiveness of ACE inhibitor, ARB, and chymase inhibitor.

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Ghrelin, identified as an endogenous ligand for the growth hormone secretagogue receptor, functions as a somatotrophic and orexigenic signal from the stomach. Ghrelin has a unique post-translational modification: the hydroxyl group of the third amino acid, usually a serine but in some species a threonine, is esterified by octanoic acid and is essential for ghrelin’s biological activities. The secretion of ghrelin increases under conditions of negative energy-balance, such as starvation, cachexia, and anorexia nervosa, whereas its expression decreases under conditions of positive energy-balance such as feeding, hyperglycemia, and obesity. In addition to having a powerful effect on the secretion of growth hormone, ghrelin stimulates food intake and transduces signals to hypothalamic regulatory nuclei that control energy homeostasis. Thus, it is interesting to note that the stomach may play an important role in not only digestion but also pituitary growth hormone release and central feeding regulation. We summarized recent findings on the integration of ghrelin into neuroendocrine networks that regulate food intake, energy balance, gastrointestinal function and growth.

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Organic anion transporters (OATs) play an essential role in the elimination of numerous endogenous and exogenous organic anions from the body. The renal OATs contribute to the excretion of many drugs and their metabolites that are important in clinical medicine. Several families of multispecific organic anion and cation transporters, including OAT family transporters, have recently been identified by molecular cloning. The OAT family consists of six isoforms (OAT1 – 4, URAT1, and rodent Oat5) and they are all expressed in the kidney, while some are also expressed in the liver, brain, and placenta. The OAT family represents mainly the renal secretory and reabsorptive pathway for organic anions and is also involved in the distribution of organic anions in the body, drug-drug interactions, and toxicity of anionic substances such as nephrotoxic drugs and uremic toxins. In this review, current knowledge of and recent progress in the understanding of several aspects of OAT family members are discussed.

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Among the 14 kinds of serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes (5-HTR), 5-HT_2C receptor (5-HT2CR) has been intensively investigated because of its physiologically and pathophysiologically important role in the brain. 5-HT2CR has been suggested to be involved in depressive disorders based on findings from pharmacological/neurochemical/behavioral studies using autopsy preparations of humans suffering from depression, animal models of depression, and animals treated with antidepressant drugs. Recently the editing of 5-HT2CR mRNA has been reported to participate in the pathogenesis of depressive disease. The RNA editing of 5-HT2CR induced by the presumable alteration of deaminase during a pathological state in depression causes changes of a base to another base (e.g., adenosine to guanosine, cytidine to uracil (thymidine)), followed by changes in amino acids constituting the second intracellular transmembrane loop that couples G proteins. Thus 5-HT2CR receptor-mediated signal transduction is changed. In the present review, the pathopharmacological significance of 5-HT2CR in special reference to RNA editing of receptors is reviewed and discussed from the aspect of development of novel therapeutics for depression.

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Recent studies have demonstrated that activation of enzymes can be observed in living cells in response to stimulation with neurotransmitters, hormones, growth factors, and so forth. Thus, the activation of enzymes was shown to be closely related to the dynamic states of various cell functions. The development of new experimental methodologies has enabled researchers to study the molecular basis of neuronal plasticity in living cells. In 1973, Bliss and his associates identified the phenomena of long-term potentiation (LTP). Since it was thought to be a model for neuronal plasticity such as learning and memory, its molecular mechanism has been extensively investigated. The mechanism was found to involve a signal transduction cascade that includes release of glutamate, activation of the NMDA glutamate receptors, Ca²⁺ entry, and activations of Ca²⁺/calmodulin-dependent protein kinases (CaM kinases) II and IV and mitogen-activated protein kinase (MAPK). Consequently, AMPA glutamate receptors were activated by phosphorylation by CaM kinase II, resulting in an increase of Ca²⁺ entry into postsynaptic neurons. Furthermore, activation of CaM kinase IV and MAPK increased phosphorylation of CREB (cyclic AMP response element binding protein) and expression of c-Fos by stimulation of gene expression. These results suggest that LTP induction and maintenance would be models of short- and long-term memory, respectively.

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The mechanisms of action of acupuncture and moxibustion as reported by Japanese researchers are reviewed. The endogenous opioid-mediated mechanisms of electroacupuncture (EA) as used in China are well understood, but these are only one component of all mechanisms of acupuncture. These studies emphasize the similarity of the analgesic action of EA to various sensory inputs to the pain inhibition mechanisms. In Japanese acupuncture therapy, careful detection of the acupuncture points and fine needling technique with comfortable subjective sensation are considered important. The role of polymodal receptors (PMR) has been stressed based on the facts that PMRs are responsive to both acupuncture and moxibustion stimuli, thermal sensitivity is essential in moxibustion therapy, and the characteristics of acupuncture points and trigger points are similar to those of sensitized PMRs. Acupuncture and moxibustion are also known to affect neurons in the brain reward systems and blood flow in skin, muscle, and nerve. Axon reflexes mediated by PMRs might be a possible mechanism for the immediate action of acupuncture and moxibustion. Reports on the curative effects of acupuncture on various digestive and urological disorders are also reviewed briefly.

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Cytokines and chemokines were originally identified as essential mediators for inflammatory and immune responses. Enhanced production and release of cytokines/chemokines are observed also in the central nervous system (CNS) under diverse pathological conditions. There is growing evidence showing that brain cytokines/chemokines play crucial roles in the neuro-glio-vascular interaction underlying the pathology of various brain disorders and therefore are potential targets for development of novel and effective therapeutics for CNS diseases. Here the evidence of the involvement of cytokines/chemokines in ischemic brain injury and pain is reviewed.

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Gabapentin, a γ-aminobutyric acid (GABA) analogue anticonvulsant, is also an effective analgesic agent in neuropathic and inflammatory, but not acute, pain systemically and intrathecally. Other clinical indications such as anxiety, bipolar disorder, and hot flashes have also been proposed. Since gabapentin was developed, several hypotheses had been proposed for its action mechanisms. They include selectively activating the heterodimeric GABA_B receptors consisting of GABA_B1a and GABA_B2 subunits, selectively enhancing the NMDA current at GABAergic interneurons, or blocking AMPA-receptor-mediated transmission in the spinal cord, binding to the L-α-amino acid transporter, activating ATP-sensitive K⁺ channels, activating hyperpolarization-activated cation channels, and modulating Ca²⁺ current by selectively binding to the specific binding site of [³H]gabapentin, the α₂δ subunit of voltage-dependent Ca²⁺ channels. Different mechanisms might be involved in different therapeutic actions of gabapentin. In this review, we summarized the recent progress in the findings proposed for the antinociceptive action mechanisms of gabapentin and suggest that the α₂δ subunit of spinal N-type Ca²⁺ channels is very likely the analgesic action target of gabapentin.

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Epilepsy is a hetergenous syndrome characterized by recurrently and repeatedly occurring seizures. Although able to inhibit the epileptic seizures, the currently available antiepileptic drugs (AEDs) have no effects on epileptogenesis. Such AEDs should be classified as drugs against ictogenesis, which are transient events in ion and/or receptor-gated channels related with triggering to evoke seizures. Epileptogenesis involves long-term and histological/biochemical/physiological alterations formed in brain structures over a long period, ranging from months to years. This review focuses on the effects of AEDs on epileptogenesis and novel candidates of antiepileptogenic drugs using a genetically defined epilepsy model animal, the spontaneous epileptic rat (SER).

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Salivary secretion occurs in response to stimulation by neurotransmitters released from autonomic nerve endings. The molecular mechanisms underlying the secretion of water, a main component of saliva, from salivary glands are not known; the plasma membrane is a major barrier to water transport. A 28-kDa integral membrane protein, distributed in highly water-permeable tissues, was identified as a water channel protein, aquaporin (AQP). Thirteen AQPs (AQP0 – AQP12) have been identified in mammals. AQP5 is localized in lipid rafts under unstimulated conditions and translocates to the apical plasma membrane in rat parotid glands upon stimulation by muscarinic agonists. The importance of increases in intracellular calcium concentration [Ca²⁺]_i and the nitric oxide synthase and protein kinase G signaling pathway in the translocation of AQP5 is reviewed in section I. Signals generated by the activation of Ca²⁺ mobilizing receptors simultaneously trigger and regulate exocytosis. Zymogen granule exocytosis occurs under the control of essential process, stimulus-secretion coupling, in salivary glands. Ca²⁺ signaling is a principal signal in both protein and water secretion from salivary glands induced by cholinergic stimulation. On the other hand, the cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase system has a major role in zymogen granule exocytosis without significant increases in [Ca²⁺]_i. In section II, the mechanisms underlying the control of salivary protein secretion and its dysfunction are reviewed.

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Fullerene (C₆₀), a condensed ring aromatic compound with extended π systems, is a novel carbon allotrope. Because of its poor solubility in polar solvents, investigation of the biological and pharmacological properties of fullerene has been difficult. Recently, water-soluble fullerene derivatives have been synthesized, and we and others have found that they have potent and selective pharmacological effects on organs, cells, enzymes, and nucleic acids. In the presence of fullerene C₆₀ derivative (10⁻⁵ M), endothelium-dependent relaxations induced by agonists in the vascular system were eliminated and acetylcholine-induced contractile response of smooth muscle was observed. Some investigators have reported free radical-scavenging activity and direct nitric oxide-quenching activity of fullerene derivatives. Knowledge of the chemical modifications, biological significance, and materials applications of functionalized fullerenes is growing rapidly; and these compounds are emerging as new tools in the field. The focus of this review is to introduce several pharmacological effects of fullerenes and to discuss the possible mechanisms of the pharmacological actions caused by previously synthesized fullerenes.

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Calcium ion (Ca²⁺) plays an important role in stimulus-response reactions of cells as a second messenger. This is done by keeping cytoplasmic Ca²⁺ concentration low at rest and by mobilizing Ca²⁺ in response to stimulus, which in turn activates the cellular reaction. The role of Ca²⁺ as a second messenger was first discovered in excitation-contraction coupling of skeletal muscle. The history of the discovery was reviewed. Characteristics of Ca²⁺ as a second messenger, diversity of target molecules, capability of rapid and massive mobilization and also of oscillatory mobilization, tendency toward localization, and on the other side, ability to cause generalized cell response were described. The possible bases for these characteristics was discussed. Ca²⁺ itself induces release of Ca²⁺ from the sarcoplasmic reticulum (Ca²⁺-induced Ca²⁺ release [CICR]). The Ca²⁺ release channel, ryanodine receptor, incorporated into lipid bilayer shows CICR activity. Ca²⁺ release induced by inositol trisphosphate also has an apparent CICR nature. The significance of CICR or apparent CICR with its inherently regenerative nature in physiological contractions of skeletal, cardiac, and smooth muscles was discussed.

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The experimental procedures to simultaneously detect contractile activity and Ca²⁺ transients by means of the Ca²⁺ sensitive bioluminescent protein aequorin in multicellular preparations, and the fluorescent dye indo-1 in single myocytes, provide powerful tools to differentiate the regulatory mechanisms of intrinsic and external inotropic interventions in intact cardiac muscle. The regulatory process of cardiac excitation-contraction coupling is classified into three categories; upstream (Ca²⁺ mobilization), central (Ca²⁺ binding to troponin C), and/or downstream (thin filament regulation of troponin C property or crossbridge cycling and crossbridge cycling activity itself) mechanisms. While a marked increase in contractile activity by the Frank-Starling mechanism is associated with only a small alteration in Ca²⁺ transients (downstream mechanism), the force-frequency relationship is primarily due to a frequency-dependent increase of Ca²⁺ transients (upstream mechanism) in mammalian ventricular myocardium. The characteristics of regulation induced by β- and α-adrenoceptor stimulation are very different between the two mechanisms: the former is associated with a pronounced facilitation of an upstream mechanism, whereas the latter is primarily due to modulation of central and/or downstream mechanisms. α-Adrenoceptor-mediated contractile regulation is mimicked by endothelin ET_A- and angiotensin II AT₁-receptor stimulation. Acidosis markedly suppresses the regulation induced by Ca²⁺ mobilizers, but certain Ca²⁺ sensitizers are able to induce the positive inotropic effect with central and/or downstream mechanisms even under pathophysiological conditions.

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Intracellular Ca²⁺ signals are important for the regulation of synaptic functions in the central nervous system. In this review, I summarize findings of our recent studies on upstream and downstream Ca²⁺ signaling mechanisms in cerebellar synapses using novel molecular imaging methods. Inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release plays a pivotal role in central synapses. The visualization of IP₃ at fine dendrites of Purkinje cells (PCs) using a fluorescent IP₃ indicator showed that intracellular Ca²⁺ concentration has a stimulatory effect on phospholipase C activity, which catalyzes IP₃ production. This indicates that metabotropic and ionotropic glutamate receptors collaborate to generate IP₃ signals. Using a novel nitric oxide (NO) indicator, the spatial distribution of NO signals originating from parallel fiber (PF) terminals was visualized. Our results show that the NO signal decays steeply with distance from the site of production in the cerebellum and is dependent on PF stimulation frequency in a biphasic manner. NO released from PF terminals generated a synapse-specific long-term potentiation of PF-PC synapse when PF was stimulated at certain frequencies. These imaging studies clarified new aspects of the regulatory mechanisms of synaptic functions.

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A dramatic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) occurs in eggs at fertilization common to all animal species examined to date, and this serves as a pivotal signal for egg activation characterized by resumption of meiotic cell division and formation of the pronuclei. In mammalian eggs, repetitive [Ca²⁺]_i rises (Ca²⁺ oscillations) each of which accompanies a propagating wave across the egg occur due to release of Ca²⁺ from the endoplasmic reticulum mainly through type 1 inositol 1,4,5-trisphosphate (IP₃) receptor. Ca²⁺ oscillations are induced by a cytosolic sperm factor driven into the egg cytoplasm upon sperm-egg fusion. A current strong candidate of the sperm factor is a novel sperm-specific isozyme of phospholipase C (IP₃-producing enzyme), PLCζ. Recent extensive research has reveled characteristics of PLCζ such as the Ca²⁺ oscillation-inducing activity after injection of PLCζ-encoding RNA or recombinant PLCζ into mouse eggs, extremely high Ca²⁺-sensitivity of the enzymatic activity in vitro, and nuclear translocation ability possibly related to cell-cycle-dependent regulation of Ca²⁺ oscillations. [Ca²⁺]_i rises cause successive activation of calmodulin-dependent kinase II and E3 ubiquitin ligase, lead to proteolysis of ubiquitinated cyclin B1 and inactivation of metaphase-promoting factor (Cdk1/cyclin B1 complex), and result in the release of eggs from meiotic arrest.

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