G proteins act as signal transducers that couple receptors to effectors through membranes. G proteins are in a GDP-bound form in the basal state, which can interact with receptors. The hormone-bound receptor promotes GDP-GTP exchange of G proteins. The GTP, Mg²⁺-bound G proteins can activate effector molecules, which in turn produce second messengers. The GTP, Mg²⁺-form is converted to the GDP-form by the GTP-hydrolysis activity of G protein α-subunits. Cl^- ions modulate the GDP-GTP cycle of G proteins. Cl^- ions, in the presence of Mg²⁺, decelerate the receptor-independent spontaneous release of GDP (ca. 6-fold by 100 mM NaCl), which will result in a lowering of the basal level of second messengers. On the other hand, the GTP-hydrolysis activity of G proteins was suppressed by 3-fold with 100 mM NaCl, which will result in keeping the G proteins in an active form for a longer time. In the absence of Mg²⁺, Cl^- ion mimics Mg²⁺ to convert GTP-bound G proteins to an active form (dissociated form), although the effects are weaker than those of Mg²⁺. These effects are very different from F^-, which is another halogen ion that interacts with G proteins.

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γ-Aminobutyric acid (GABA) directly inhibits the postsynaptic membrane through GABA_A receptor Cl^- channel complexes. This inhibition is promoted or blocked by various intra and extracellular substances at the site of either receptor or channel. Recent studies focused on the intracellular inhibitory mechanisms of the GABAergic response in neurons : one is the inhibition of GABA receptors by the increase in intracellular Ca²⁺ and the other is inhibition through ATP receptors triggered by the decrease in intracellular ATP level. In addition, the spontaneous inhibitory postsynaptic currents (IPSCs) induced by GABA released from the nerve terminals were suppressed by activation of the GABA_B receptor, which acts as a negative autoreceptor in the nerve ending. The intracellular mechanism of the suppression will be discussed.

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Glycine is a major inhibitory neurotransmitter in the spinal cord and brain stem. Glycine acts by increasing the Cl^- permeability through activation of a specific receptor/ion channel complex consisting of a pentameric subunit assembly. Molecular cloning has disclosed the nature of receptor subunits α and β. While the role of the β subunit is still unclear, the α subunit functions in both ligand (agonist/antagonist) binding and ion channel formation. It has been demonstrated that there are two isoforms of the α subunit, α₁ and α₂. The mRNAs encoding these subunit isomers are transcribed from different genes, in spite of their structural similarity. The α₁ mRNA is abundant in adult spinal cord, whereas the α₂ mRNA is mainly expressed in developing spinal cord as well as various regions of brain tissue. The single channel properties were examined in outside-out patches excised from Xenopus oocyte membrane expressing α₁ or α₂ homomeric receptors. The mean open time of α₂ channels was 70-times longer than that of α₁ channels. The subunit switching from α₂ to α₁, and resulting shortening of channel kinetics, may ensure a rapid motor control in adult animals.

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Recently, several lines of evidence have indicated the important roles of glial cells, especially astrocytes, in the regulation of neuronal functions. The neuron-glia interaction is one of the most important issues in neuroscience, including neuropharmacology. I reviewed the present status and perspectives on the physiologic and pathologic functions of astrocytes in relation to the roles of intracellular Cl^-. Astrocytes have different types of Cl transport systems, such as voltage-sensitive and ligand-gated channels; HCO₃^--C1- exchange; and Na⁺, K⁺, Cl^- cotransport systems. Anion exchange and cotransport systems are responsible for intracellular pH regulation and astrocytic volume regulation, respectively. Especially, astrocytic volume regulation is physiologically important for reducing the concentrations of K⁺ and glutamate in the extracellular space by their uptake systems. Disturbance of astrocytic volume regulation is expressed as astrocytic swelling, which is usually observed in various brain pathologic states including ischemia. Experimentally, glutamate caused a typical swelling of astrocytes in culture by Cl^- and Ca⁺⁺-dependent processes. Glutamate-induced swelling is qualitatively different from reversible swelling induced by hypoosmotic medium. Recently, we found that Cl^- is intracellular factor for modulating the receptor-adenylate cyclase system in brain slices. Similarly, the receptor and forskolinstimulated adenylate cyclase of astrocytes showed a clear Cl^- dependence. This was functionally confirmed by astrocytic morphological transformation induced by the cyclic AMP system.

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Intracellular chloride ion concentration ([Cl^-]_i) plays an important role in cellular functions including the control of membrane potential and excitability. In neurons, Cl equilibrium potential (E_Cl) is lower or higher than the membrane potential (E_m), suggesting that [Cl^-]_i is lower or higher than that expected from passive distribution. As the mechanisms to control [Cl^-]_i, active outwardly or inwardly directed Cl^- transport systems have been reported. The former includes Na⁺-dependent Cl^-/HCO₃^- exchanger, K⁺/Cl^- cotransporter and ATP-dependent Cl^- pump; and the latter includes Na⁺/K⁺/2Cl^- cotransporter and amino acid-dependent Na⁺/Cl cotransporter. In hippocampal pyramidal cells, recent studies using a Cl^--sensitive fluorescent probe to monitor [Cl^-]_i revealed the presence of an ATPdependent Cl^- pump and a Na⁺/K⁺/2Cl^- contransporter, and an uneven distribution of [Cl^-]_i (cell body<dendrite) and these Cl^- transport systems. Intracerebroventricular administration of an inhibitor of the ATP-dependent Cl^- pump, ethacrynic acid, induces status epilepticus in mice. Thus, it appears to be necessary to elucidate cellular and molecular mechanisms of Cl^- transporters and their control systems for a better understanding of Cl^--related functions in neurons.

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To selectively isolate hepatocytes from the periportal (PP) and perivenous (PV) regions of rat liver acinus, we compared two different perfusion methods with collagenase: the bidirectional perfusion method (2-P) and the one way perfusion method (1-P). We determined the optimal conditions for each method on the basis of the zonal selectivity of isolated hepatocytes with a hematoxylin-eosin stained liver specimen. By both methods, hepatocytes were selectively isolated from the PP and PV regions. Comparing cell yield and cell viability after the two perfusion methods, 1-P was found to be better than 2-P. Density gradient centrifugation with Percoll was found to be an effective procedure for removing the damaged hepatocytes. We concluded that 1-P could isolate viable PP and PV hepatocytes with a normal glucagon-cyclic AMP response and ultrafine structure in high yield from rat liver.

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Changes in β-adrenoceptors, GTP binding (G) proteins and adenylate cyclase (AC) activity of ventral prostates of rats during aging were studied in these experiments. The density of β-adrenoceptors increased markedly after birth, reaching a maximum in the tissues at 16 weeks of age and remained at this level for about 90 weeks. When stimulated by isoproterenol, the AC activity increased 4 and 22-fold in 2 and 8-week-old rats, respectively, but only 7-fold in both 16 and 104-week-old rats. Activation of AC by forskolin was the greatest during the 2 weeks after birth and then showed a sharp decrease, reaching a plateau in the tissues at 8 weeks. B_max value of (³⁵S)GTPγS binding to the tissues was the largest at 8 weeks. Pretreatment of the tissues with pertussis or cholera toxin caused age-dependent changes in both the binding abilities of Gi and Gs proteins to GTP that coincided with changes in the binding of G proteins to GTP. There was no difference between the abilities of Gi and Gs proteins to bind to GTP in the tissues of rats of the respective age. These results show that changes in the binding ability of G proteins to GTP influence the function of ventral prostates of rats during the aging process, and this is mediated through the regulation of AC activity.

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We studied the healing promoting action of Z-103 on the chronic gastric ulcer induced by acetic acid (AAU) or Fe-ascorbic acid (FAU) in rats. The area of the gastric ulcers, hydroxyproline (Hyp) and DNA contents in the ulcer region were measured as an index of ulcer healing. The area of gastric ulcers was the largest on day 4 and thereafter gradually decreased, but the ulcers still remained at the 14th day. Hyp contents in the ulcer region decreased until the 7th day in both models, and then this level increased. Significant decrease in DNA contents in the ulcer region was observed on the 7th day only in FAU. In AAU and FAU, administration of Z-103 (3 mg/kg/day×2, p.o.) resulted in a significant decrease in the area of gastric ulcers on the 14th day and a significant increase in Hyp contents in the ulcer region on the 7th day as compared with the control group. Z-103 increased the DNA contents in the ulcer region on the 4th day in AAU and on the 7th day in FAU. These results suggest that tissue destruction surrounding the ulcer region in AAU and FAU models might occur until the 4th or 7th day after operation, and that the acceleration of ulcer healing by Z-103 on these models may be facilitated by the wound healing action of this drug.

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It has been presumed that a primary site of termination of the baroreceptor afferent was in the nucleus tractus solitarii (NTS). The intermediate region of the nucleus, located at the level of the obex, contains a large number of catecholamine neurons. The principal noradrenergic innervation of the NTS appears to arise from neurons of the so-called A2 group. It has been proposed that catecholamines released by A2 neurons serve to lower blood pressure and heart rate. We performed experiments to determine the effects of α-adrenoceptor agonists and antagonists on the baroreceptor reflex and alpha-adrenoceptor. Rabbits anesthetized with chloralose-urethane were injected with an alpha-adrenoceptor agonist, such as noradrenaline, phenylephrine or clonidine, into the NTS. An adrenoceptor-antagonist, such as yohimbine, phentolamine, prazosin or corynanthine was injected into the NTS of other anesthetized rabbits. Clonidine markedly lowered the blood pressure and heart rate, and it inhibited the baroreceptor reflex responses. Noradrenaline or phenylephrine did not lower the blood pressure and heart rate and scarcely inhibited the baroreceptor reflex. Yohimbine remarkedly inhibited the baroreceptor reflex responses; on the other hand, prazosin, corynanthine and phentolamine produced little inhibition of the responses. It is concluded that there are α₁ and α₂-receptors in the NTS of the rabbits, and the α₂-receptors play an important role in the neurotransmission in the NTS.

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We performed these studies to determine whether sialic acid (SIA) existed in the inflammatory exudate of the carrageenin (Car) -air pouch model and to elucidate the mechanisms of the antiinflammatory action of SIA on the Car-induced edema in rat hind paws. SIA (113.20 ± 10.73 μg/ml) was detected in the exudate of Car-air pouch, and the plasma SIA (660.29 ± 29.38 μg/ml) in Car-air pouch rats was significantly higher than that (490.00 ± 29.37 μg/ml) in control rats. SIA (300 mg/kg, s.c.) suppressed the delayed phase of Car-induced edema, and it also suppressed the edema induced by Car plus arachidonic acid, Car plus PGE₂, and bradykinin plus PGE₂. However, SIA did not affect the edema induced by dextran, histamine, bradykinin, and Car plus PGE₁. SIA affected neither the PG production in rats nor the (³H)PGE₂-receptor binding of guinea pig ileum, and SIA reduced the PGE₂-induced contraction of isolated guinea pig ileum. The above results suggest that SIA induces the antiinflammatory effects via its antagonism against PGE₂. Furthermore, the presence of SIA in the inflammatory exudate and the higher concentration of SIA in the plasma than in the exudate might suggest that SIA plays patho-physiologically protective roles in inflammatory states.

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We have investigated the effect of the NO synthesis inhibitor L-N^G-nitro arginine (L-NNA) on the NANC relaxation induced by electrical field stimulation (FS) or the 5-HT₁-like receptor agonist 5-carboxamidotryptamine (5-CT) in the longitudinal muscle of the guinea pig proximal colon. In the presence of atropine (0.2 pM) and guanethidine (5. 0 μM), NANC relaxations induced by FS (0. 1 ?? 10 Hz, 160 mA, 0.3 ms) or 5-CT (1.0-30 μM) were completely abolished by tetrodotoxin (0. 3 μM). LNNA (10 ?? 100 μM) produced concentration-dependent inhibition of NANC relaxations, but D-NNA had no effect. This inhibitory effect of L-NNA on NANC relaxations was prevented by L-arginine (1 mM) or L-citrulline (1 mM), but was not affected by D-arginine (1 mM). Like L-NNA, L-NMMA (100 ?? 300 μM) also inhibited the NANC relaxation induced by FS and 5-CT. These results suggest that nitric oxide (NO) or a substance releasing NO may contribute to NANC relaxations of guinea pig proximal colon induced by FS and the stimulation of 5-HT₁-like receptors.

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