GABA receptors are classified into two receptor subtypes: GABAA and GABAB receptors. The GABAA receptor, one of the ionotropic type receptors, is formed by various subunits (α, β, γ and δ subunits) and constitutes the GABA-gated Cl- channel. The different combinations of these subunits are known to produce functionally heterogeneous GABAA receptors both pharmacologically and physiologically. On the other hand, GABAB receptor is known to be metabotropic type which is negatively coupled with adenylate cyclase and inositol phosphate turnover systems via inhibitory GTP binding protein.
The muscarinic acetylcholine receptor (mAChR) is an integral membrane protein that transduces stimulus to effectors through the activation of guanine nucleotide-binding (G) proteins. Four or more subtypes of mAChR were detected in various tissues, and their primary structures were elucidated by cloning and sequence analysis of complementary DNA. Functional differences between them existed when they were expressed in clonal culture cells. mAChRI (m1) and mAChRIII (m3) preferentially activated phosphoinositide (PI) hydrolysis and opened Ca2+-activated K+ channels followed by closure of the M (K+)-currents, while such current activities were rarely evoked by mAChRII (m2) and mAChRIV (m4)-transformed cells. Although it has been reported that mAChRII and mAChRIV inhibited adenylate cyclase, there was little or no such inhibition by mAChRI and mAChRIII. It is known that heart and neuronal mAChR modulate voltage-sensitive Ca2+ currents, but which species of mAChR subtypes are involved has been poorly understood. Recently we identified that endogenous mAChRIV and exogenous mAChRII expressed in NG108-15 neuroblastoma-glioma hybrid cells, but not mAChRI and mAChRIII, efficiently depressed high-threshold Ca2+ currents in a pertussis toxin-sensitive manner.
Calcium ion (Ca2+) is considered to be involved in the regulation of numerous cellular processes. CaM kinase II is present at the highest concentration in the brain and is considered to be involved in the regulation and coordination of numerous cellular processes. CaM kinase II is activated by Ca2+ /calmodulin and simultaneously undergoes autophosphorylation. It has not been determined whether the enzyme is activated in the cell systems in response to the increase in cytoplasmic Ca2+ concentration. We have studied CaM kinase II in several kinds of cells including the primary cultures of cerebellar granule cells and the cell lines of rat embryo fibroblast 3Y1 cells, neuroblastoma cells, PC12 cells and C6 glioma cells. The immunohistochemical analysis demonstrated the presence of CaM kinase II in all of the cells examined. Furthermore, the kinase in cerebellar granule cells was activated by the stimulation of the glutamic acid receptor. Autophosphorylation of CaM kinase II in 3Y1 cells was stimulated by the addition of growth factors. These results suggest that CaM kinase II undergoes activation and autophosphorylation in response to various stimuli to the cells and is regulated in the dynamic state.
Morphine and ethanol drugs known to develop tolerance and dependence, induce changes in the adenylate cyclase system. Morphine inhibits the adenylate cyclase activity in NG 10 8-15 cells and causes increases in adenylate cyclase synthesis and the down-regulation of opiate receptors in cells treated for several days. Chronic exposure of NG108-15 cells to ethanol also causes a decrease in the mRNA of the GTP-binding protein (Gs). These observations suggest the possibility that a group of genes is expressed in response to morphine or ethanol during the acquisition of tolerance and dependence. Recently, it has been reported that cAMP regulates a number of genes through a cAMP response element (CRE) in their promotor regions and that nuclear CRE-binding proteins bind specifically to the CRE to stimulate the transcription of cAMP-responsive genes. The gel shift assay with a single stranded oligo-DNA of CRE in a somatostatin promotor region was employed to examine the possibility of transcriptional regulation of cAMP-inducible genes by chronic morphine or ethanol treatment of NG 108-15 cells. When the nuclear proteins from the cells treated with morphine or ethanol for several days were provided for the assay, the amounts of DNA-protein complex were decreased. The decreased complexes were recovered by 1 ?? 2 days after morphine withdrawal. The nuclear proteins were purified partially by a combination of chromatography on Q-Sepharose, Sephacryl S-300 and DNA affinity-Sepharose. Changes in CRE-binding proteins from the cells treated chronically with morphine or ethanol suggest that these drugs can modulate the expression of cAMP-inducible genes through which tolerance and dependence may develop.
An increase in inositol 1, 4, 5-trisphosphate (IP3) formation in rat mast cells precedes an elevation in intracellular Ca2+ levels, which triggers the process(es) leading to histamine release. By means of a transmission electron microscope, it was revealed that when permeabilized mast cells were exposed to potassium antimonate, antimonate precipitates in the endoplasmic reticulum (ER) in the form of calcium antimonate, indicating that the ER is the intracellular Ca store in rat mast cells. IP3 at concentrations higher than 0.5 μM preferentially releases Ca2+ from the isolated ER of mast cells. GTP was also effective in releasing Ca2+ from the ER. IP3-induced Ca2+ release was inhibited by pretreatments with cAMP and antiallergic drugs. An increase in the intracellular Ca2+ concentration may lead to an activation of calmodulin, C kinase and cytoskeletal elements in sequence. Furthermore, microtubules may play an important role in the process (es) leading to Ca2+ release from the intracellular Ca store and subsequent histamine release, without affecting IP3 formation. In contrast, microfilaments seem to participate not only in the extrusion but also in the reincorporation of the mast cell granules, having no influence on intracellular Ca2+ release. Substance P (SP) is one of the most effective neuropeptides for releasing histamine from mast cells. Structure-activity relationship studies indicate that basicity at the N-terminal and hydrophobicity at the C-terminal are requisite for its histamine releasing activity. SP effectively released Ca2+ from the intracellular Ca store. The site of action of SP on the mast cell surface seems to be the same as that of compound 48 /80. Eosinophil major basic protein (MBP) and histone are also effective for releasing histamine. The cDNA sequences of two subclasses of guinea pig MBP have been determined. These proteins may be released at the site of inflammation from the cells activated by the chemical mediators released from mast cells, and consequently, mast cell activation was reinforced. Such cell-to-cell interaction may be the reason for the augmentation of inflammation.
The effect of palytoxin (PTX) on catecholamine (CA) secretion from cultured bovine adrenal chromaffin cells was examined. PTX (> 10-10 M) induced CA secretion concentration-dependently. About 40 ?? 50% of the total cellular CA was secreted during a 20 min incubation with 3 × 10-8 M PTX. PTX caused increases in [22Na]+ and [95Ca]2+-influxes into the cells, which were not affected by TTX. PTX-induced CA secretion and [22Na]+ and [45Ca]2+-influxes were significantly inhibited by quinidine and aprindine, antiarrythmic drugs. Ca2+-channel blockers such as nifedipine, verapamil, Co2+, and Cd2+ inhibited both CA secretion and [45Ca]2+-influx induced by PTX. These results indicated that PTXinduced CA secretion was mediated by activation of Na+-dependent, TTX-insensitive voltage-dependent Ca2+-channels. PTX-induced [22Na]+-influx was inhibited by amiloride, an inhibitor of the Na+-H+ exchange system, suggesting that the Na+-H+ exchange mechanism might be involved in PTX-induced [22Na]+-influx into the cells. The effects of flavonoids on CA secretion from permeabilized adrenal chromaffin cells were examined. CA secretion from the cells in response to a direct Ca2+ challenge was inhibited by quercetin (> 10-5 M) and apigenin (> 10-5 M). These flavonoids also inhibited phorbol ester TPA-induced CA secretion. Therefore, the inhibitory effects of flavonoids on CA secretion were thought to be attributed to their inhibitory effects on PKC.
The intracellular concentration of Ca2+([Ca2+]i) in eukaryotic cells is low under resting conditions. Membrane stimulation can produce a transient increase in [Ca2+]i from submicromolar to micromolar levels. Classically, the effects of elevated [Ca2+]i on eukaryotic cell responses are excitatory: they induce muscle contraction or activate metabolic systems. However, micromolar concentration of Ca2+ has been found to inhibit the actin-myosin-ATP interaction of the lower eukaryote Physarum polycephalum, which is the first example of an effect transiently abolished by elevation of [Ca2+]i in response to extracellular stimulation (Kohama, K.: Trends Pharmacol. Sci. 22, 433-435, 1991). The inhibitory effects on the interaction are closely related to myosin phosphorylation. The activities of kinases to phosphorylate myosin was also inhibited by Ca2+. This article describes the inhibitory mode for Ca2+ regulation of the actomyosin system in Physarum and reviews this inhibitory mode discovered subsequently in many different cell types in both plants and animals.
Characteristics of the ATP release from non-neuronal tissues were reviewed in connection with that from neuronal tissues. α1-Adrenoceptor and M3-cholinoceptor stimulation produced a postjunctional ATP release from smooth muscles such as the vas deferens and ileal longitudinal muscles of the guinea pig, suggesting the existence of a coupling mechanism between the stimulation of the receptor for the transmitter and postjunctional ATP release. Accompanied with positive inotropic action, cardiotonics elicit postjunctional ATP release, which is derived from mitochondria, in atrial muscles, whereas these drugs are incapable of producing the release from papillary muscles. Possible physiological roles of ATP released to synapses from nonneuronal tissues are considered as follows: 1) the nucleotide may act as an opener of Ca2+ and K+ channels, 2) ATP in the synapse seems to serve as a transynaptic neuromodulator after conversion to adenosine.
Cells are equipped with complex mechanisms for synthesis of ATP and try to keep the intracellular level of this compound, which is indispensable for maintenance of normal function and integrity, as constant as possible. Thus, it is generally believed that ATP rarely cross the plasma membrane of viable cells. However, since the first report of Holton in 1959, the release of nucleotides from cells has become an established fact, and the physiological role and metabolism of the released ATP has become an important topic; potent actions of extracellular purine nucleotides and nucleosides have been recognized for many years. In 1972, Burnstock demonstrated that ATP has a transmitter role in certain types of nerves and proposed a concept of purinergic nerve. The receptor for purine nucleotides, designated as P2, as opposed to P1, by Burnstock in 1978 was further subclassified in 1985 into P2X and P2Y by himself, and we now have a train of P2 receptors, such as P2S, P2T, P2Z and so forth. In this review, I summarized the characteristics of these purinoceptors. Pharmacological effects and metabolism of extracellular nucleotides were discussed and a brief mention was made of ecto-nucleotidases.