Calcium channel gamma subunits provide insights into the evolution of this gene family.

The gamma subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct gamma subunits have been described from human and/or mouse. The first identified member of this group of proteins, gamma(1), is a component of the L-type calcium channel expressed in skeletal muscle. A second member, gamma(2), identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel gamma subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian gamma subunits suggests the following relationship ((((gamma(2), gamma(3)), (gamma(4), gamma(8))), (gamma(5), gamma(7))), (gamma(1), gamma(6))) that indicates that they evolved from a common ancestral gamma subunit via gene duplication. Our analysis reveals that the novel gamma subunit gamma(6) most closely resembles gamma(1) and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other gamma subunits. Rat gamma subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of gamma(1) mRNA and the long isoform of gamma(6) mRNA is most robust in skeletal muscle, while gamma(6) is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the gamma subunits, we propose that gamma(1) and gamma(6) may share common physiological functions distinct from the other homologous gamma subunits.

Transforming growth factor-beta 1 promotes re-elongation of injured axons of cultured rat hippocampal neurons.

Transforming growth factor-beta 1 (TGF-beta 1) is highly expressed in neural tissues following brain injuries. To determine a role of TGF-beta 1 in neural pathologies, axons of cultured rat hippocampal neurons were cut by local irradiation of laser beam, and the effect of TGF-beta 1 on neurite regeneration following axotomy was investigated. The axonal growth was stopped by laser irradiation, but addition of TGF-beta 1 remarkably promoted the axonal re-elongation from the injured site. The effect of TGF-beta 1 was concentration dependent and seen maximally at a concentration of 1 ng/ml. These results suggest that TGF-beta 1 has a capability of promoting axonal regeneration of brain neurons after lesioning.

An herbal prescription, S-113m, consisting of biota, ginseng and schizandra, improves learning performance in senescence accelerated mouse.

The effect of an herbal prescription, S-113m, consisting of biota, ginseng and schizandra, on learning and memory performance was studied in the senescence accelerated mouse (SAM). A solid diet containing 1% (w/w) S-113m was given to SAM from 1 month of age. A behavioral experiment, started 4 or 9 months later, revealed prominent learning impairment in SAMP8, a senescence accelerated-prone mouse. Chronic ingestion of S-113m improved the memory retention disorder of SAMP8 in a passive avoidance test and increased the conditioned avoidance rate in a lever-press test at the age of 10 months. The preparation also facilitated the memory retention deficit in the passive avoidance test in 10-month-old SAMR1, a senescent resistant substrain. These results raise the possibility that S-113m might be useful for treating physiological aging and age-related memory deficits in human.

Antagonistic effect of N-(3-Aminopropyl)cyclohexylamine on neurotrophic action of spermine in primary cultured rat hippocampal and cerebellar neurons.

We previously found that spermine potently promotes the neuronal survival and regeneration of primary cultured brain neurons. N-(3-Aminopropyl)cyclohexylamine (APCHA) was originally developed as a spermine synthase inhibitor. To test if endogenous spermine biosynthesis contributes to neuronal survival and morphogenesis, we examined the effects of APCHA in primary cultured rat hippocampal and cerebellar neurons. APCHA at concentrations up to 10(-6) M did not affect the neuronal survival, but significantly blocked the survival-promoting effect of spermine (10(-8) M). APCHA also blocked the spermine-induced promotion of neurite regeneration following axotomy. Unlike APCHA, another cyclohexylamine derivative trans-4-methylcyclohexylamine did not affect the neurotrophic effect of spermine. These results suggest that in primary cultured brain neurons, APCHA works as a spermine antagonist rather than as a spermine synthesis inhibitor.

Beneficial effects of biota, a traditional Chinese herbal medicine on learning impairment induced by basal forebrain-lesion in mice.

The effects of biota (Biota orientalis ENDL.) on learning impairment produced by bilateral lesion of basal forebrain in mice were studied using step-through and step-down type passive avoidance tasks. Basal forebrain-lesion was generated by applying radiofrequency current. Behavioral experiment, started 15d after the survey, revealed prominent delay in the memory acquisition process in the lesioned mice. The operation also induced memory retention deficit in both learning tests. Chronic oral administration of ethanol extract of biota seeds at doses of 250 and 500 mg/kg/d, from the day of surgery until the end of the behavioral test, dose-dependently improved memory acquisition impairment in the step-down test and memory retention disturbance in both behavioral tasks. The preparation also had a tendency to alleviate memory acquisition impairment in the step-through test. Although precise action mechanisms of biota extract remains speculative, such as what component works on which target, the present results clearly suggest that the preparation affects the learning and memory processes in the central nervous system and improves the impairment of memory acquisition and retention disturbances produced by basal forebrain-lesion.

Polyamines promote regeneration of injured axons of cultured rat hippocampal neurons.

Axons of cultured rat hippocampal neurons were injured by local irradiation of laser beam, and the effects of spermine, spermidine and putrescine on neurite regeneration following axonal injury were investigated. The axonal growth was stopped by laser irradiation, but addition of spermine remarkably promoted the axonal re-elongation from the injured site. Spermine affected neither the neurite branching at proximal part of injured axons nor the growth of uninjured dendrites. The effect of spermine was concentration dependent and seen maximally at a concentration of 10(-8) M. Spermidine and putrescine also promoted the axonal re-elongation in a concentration-dependent manner. The effects of three polyamines were very similar, and no additivity was observed when maximally effective concentrations of polyamines were added together, suggesting that they act through a common mechanism. Unlike polyamines, basic fibroblast growth factor (bFGF) did not promote the axonal re-elongation from the injured site, but rather stimulated the formation of axonal branches at proximal part of injured axons, supporting that the promotion of axonal re-elongation is a specific action of polyamines. Concomitant addition of spermine and bFGF additively or synergistically promoted both the axonal re-elongation from the injured site and the branch formation at proximal part of injured axons. These data suggest that polyamines have a capability of promoting axonal regeneration of brain neurons after lesioning.

Structural requirement for neurotrophic activity of spermine in cultured rat hippocampal neurons.

We investigated the structure-activity relationship for the neurotrophic activity of spermine by comparing the effects of several synthetic spermine analogues on the survival of cultured rat hippocampal neurons. N,N'-bis(3-aminopropyl)-1,6-hexanediamine and N,N'-bis(3-aminopropyl)-1,3-propanediamine did not promote the neuronal survival, suggesting that the central butanediamine structure is essential for the neurotrophic activity. Furthermore, N1,N12-bis(ethyl)spermine significantly promoted the neuron survival, but its maximum effect was smaller than that of spermine, indicating that two terminal primary amines are required for full agonist activity. The structural requirement for the neurotrophic activity of spermine was different from those for any other reported activities of polyamines.

Prolongation of life span and improved learning in the senescence accelerated mouse produced by aged garlic extract.

The effects of aged garlic extract (AGE) on longevity and learning and memory performances were studied in the senescence accelerated mouse (SAM). A solid diet containing 2% (w/w) AGE was given to SAM from 2 months of age. The survival ratio of SAM P8, senescence accelerated animals, treated with AGE was significantly higher than that of untreated controls. AGE, however, did not affect the life span of SAM R1, a senescence-resistant strain. AGE had no effect on body weight and motor activity. In the passive and conditioned avoidance tests, AGE markably improved a memory acquisition process in the step-down and shuttle-box tests, and also a retention process in the step-through and step-down tests in SAM P8. The beneficial effects of AGE were observed in a memory retention process in the step-down test and in an acquisition stage in lever-press test in SAM R1. These results suggest the possibility that AGE might be useful for treating physiological aging and age-related memory deficits in humans.

N-(3-aminopropyl)-cyclohexylamine blocks facilitation by spermidine of N-methyl-DL-aspartate-induced seizure in mice in vivo.

The facilitating or antagonizing effects of polyamine analogues on N-methyl-DL-aspartate (NMDLA)-induced seizures were investigated using mice. Intracerebroventricular injection of spermidine and spermine, but not putrescine, shortened the latency to appearance of clonic convulsion induced by subcutaneous administration of NMDLA. Injection of N-(3-aminopropyl)cyclohexylamine (APCHA) alone did not affect the NMDLA-induced seizure. However, APCHA, when administered together with spermidine, clearly antagonized the facilitating effect of spermidine on the NMDLA-induced seizure. Another cyclohexylamine derivative, trans-4-methylcyclohexylamine, did not block the effect of spermidine. APCHA also antagonized the facilitation by D-serine of NMDLA-induced seizure, although the blocking effect for D-serine was weaker than that for spermidine. APCHA should be useful as a new tool for pharmacological studies on the neuromodulatory action of polyamines.

Polyamines promote neurite elongation of cultured rat hippocampal neurons.

The effects of spermine, spermidine and putrescine on the survival and morphology of hippocampal neurons obtained from rat embryos were investigated using low cell density culture. The number of surviving neurons gradually decreased in the control cultures, while addition of spermine significantly promoted neuronal survival. The survival-promoting effect of spermine was seen maximally at a concentration of 10(-8) M. On the other hand, spermidine and putrescine showed no significant effect on neuronal survival. The axon length of neurons cultured in the presence of spermine was significantly longer than that in the medium with no added spermine, indicating that spermine promotes process elongation. The concentration-effect curve for spermine was bell-shaped with the maximum effect at a concentration of 10(-8) M. However, the number of processes per soma, the elongation of dendrites and the process branching in axons were not significantly affected by the presence of spermine. Addition of spermidine and putrescine also promoted the axon elongation of cultured neurons, without affecting the branching number. The effects of spermidine and putrescine on neuronal growth were very similar to that of spermine in terms of the effective concentrations and the patterns of morphological changes. Since spermine specifically promoted neuronal survival while three polyamines affected the neuronal morphology in a similar manner, it is possible that the effects of polyamines on neuronal survival and neurite elongation are mediated by different mechanisms.

The adenosine analogue N6-L-phenylisopropyladenosine inhibits catecholamine secretion from bovine adrenal medulla cells by inhibiting calcium influx.

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.