Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [3H]saxitoxin ([3H]STX) binding in a time‐dependent manner (t1/2 = 26 h). Insulin (100 nM for 4 days) increased the Bmax of [3H]STX binding by 49% without changing the KD value and also augmented the maximal influx of 22Na+ due to 560 µM veratridine by 39% without altering the EC50 value of veratridine. The stimulatory effect of insulin on 22Na+ influx was concentration‐dependent with an EC50 of 3 nM, whereas insulin‐like growth factor (IGF)‐I had little effect at 1 nM. Ptychodiscus brevis toxin‐3 allosterically potentiated veratridine (100 µM)‐induced 22Na+ influx by approximately twofold in both insulin‐treated cells and untreated cells. Veratridine‐induced 45Ca2+ influx via voltage‐dependent Ca2+ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine‐induced 22Na+ influx via the nicotinic receptor‐ion channel complex and high‐K+ (direct activation of voltage‐dependent Ca2+ channels)‐induced 45Ca2+ influx. Stimulatory effects of insulin on [3H]STX binding and veratridine‐induced 22Na+ influx were nullified by simultaneous treatment with either 5,6‐dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na+ channel α‐subunit. These results suggest that the binding of insulin to insulin (but not IGF‐I) receptors mediates the up‐regulation of functional Na+ channel expression at plasma membranes; this up‐regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).

Our previous [3 H]saxitoxin binding and 22 Na influx assays showed that treatment of cultured bovine adrenal chromaffin cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), decreased the number of cell surface Na channels (IC 50 ϭ 19 nM) but did not alter their pharmacological properties; Na channel downregulation developed within 3 h, reached the peak decrease of 53% at 15 h, and was mediated by transcriptional/translational events. In the present study, treatment with 100 nM TPA lowered the Na channel ␣-subunit mRNA level by 34 and 52% at 3 and 6 h, followed by restoration to the pretreatment level at 24 h, whereas 100 nM TPA elevated the Na channel ␤ 1 -subunit mRNA level by 13-61% between 12 and 48 h. Reduction of ␣-subunit mRNA level by TPA was concentration-dependent (IC 50 ϭ 18 nM) and was mimicked by PDBu but not by the biologically inactive 4␣-TPA; it was prevented by H-7, an inhibitor of PKC, but not by HA-1004, a less active analogue of H7, or by H-89, an inhibitor of cyclic AMPdependent protein kinase. Treatment with cycloheximide, an inhibitor of protein synthesis, per se sustainingly increased the ␣-subunit mRNA level and decreased the ␤ 1 -subunit mRNA level for 24 h; also, the TPA-induced decrease of ␣-subunit mRNA and increase of ␤ 1 -subunit mRNA were both totally prevented for 24 h by concurrent treatment with cycloheximide. Nuclear run-on assay showed that TPA treatment did not alter the transcriptional rate of the ␣-subunit gene. A stability study using actinomycin D, an inhibitor of RNA synthesis, revealed that TPA treatment shortened the t 1/2 of ␣-subunit mRNA from 18.8 to 3.7 h. These results suggest that Na channel ␣-and ␤ 1 -subunit mRNA levels are differentially downand up-regulated via PKC; the process may be mediated via an induction of as yet unidentified short-lived protein(s), which may culminate in the destabilization of ␣-subunit mRNA without altering ␣-subunit gene transcription. Key Words: Sodium channel ␣-and ␤ 1 -subunits-Protein kinase C-Down-and up-regulationsNorthern blot-Nuclear run-on assay-mRNA stability.

Treatment of cultured bovine adrenal chromaffin cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), an activator of protein kinase C (PKC), decreased [3H]saxitoxin ([3H]STX) binding in a concentration (IC50 = 19 nM)‐ and time (t1/2 = 4.5 h)‐dependent manner. TPA (100 nM for 15 h) lowered the Bmax of [3H]STX binding by 53% without altering the KD value. Phorbol 12,13‐dibutyrate (PDBu) also reduced [3H]STX binding, whereas 4α‐TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H‐7 (an inhibitor of PKC), but not H‐89 (an inhibitor of cyclic AMP‐dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine‐induced 22Na+ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine‐induced 22Na+ influx by brevetoxin. These results suggest that an activation of PKC down‐regulates the density of Na channels without altering their pharmacological features; this down‐regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.

No abstract

This study investigated the role of human T-cell lymphotropic virus type I HTLV-I infection in 11 patients who developed slowly progressive myelopathy with abnormal spinal cord lesions. The authors performed clinical and neuroradiological examinations and calculated the odds that an HTLV-I-infected individual of a specific genotype, age, and provirus load has HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Anti-HTLV-I antibodies were present in both the serum and cerebrospinal fluid in all of the patients. Abnormal magnetic resonance imaging (MRI) lesions were classified as cervical to thoracic type (CT type), cervical type (C type), and thoracic type (T type). In each type, there was swelling of the spinal cords with high-intensity lesions, which were located mainly in bilateral posterior columns, posterior horns, or lateral columns. Virological and immunological analyses revealed that all patients showed a high risk of developing HAM/TSP. These 11 patients may have developed HAM/TSP, as manifested by spinal cord abnormalities shown on MRI. These MRIs implicate clinical variability of HAM/TSP, which may indicate active-early stages of HAM/TSP lesions.