The possible participation of cyclic AMP in the stress‐induced synthesis of two small stress proteins, hsp27 and αB‐crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB‐crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β‐adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite‐ or heat‐induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB‐crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB‐crystallin showed that the modulation of the stress‐induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB‐crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB‐crystallin is suppressed.

The two small stress proteins, HSP27 and alpha B crystallin, are expressed widely in normal rat tissues and abundantly in skeletal muscle. In order to clarify the physiological significance of these stress proteins, the changes in their levels were determined immunochemically, in the slow-twitch soleus muscle and fast-twitch extensor digitorum longus muscle or rectus femoris muscle of growing rats, and in those of adult rats during denervation and tenotomy. HSP27 was quantitated by specific immunoassay, similar to that for alpha B crystallin, with antibodies raised in rabbits against purified rat HSP27. In adult rats, HSP27 was present at high levels in tissues composed of striated muscle, and it was present at much higher levels in the soleus muscle than in the rectus femoris or extensor digitorum longus muscle, as is alpha B crystallin. However, in rats of perinatal age (from prenatal day 2 to postnatal day 3), levels of HSP27 in the rectus femoris muscle were enhanced like those in the soleus muscle, reaching the maximum levels at postnatal day 3. Thereafter HSP27 in the fast-twitch muscle showed a steep decrease. The increase in alpha B crystallin in the hindlimb muscles was also observed in the perinatal period. However, alpha B crystallin concentrations in the soleus muscle of perinatal rats were as low as those in rectus femoris muscle. The transection of the sciatic nerve resulted in decreases in the levels of HSP27 and alpha B crystallin in the soleus muscle of adult rats, together with increases in the levels of the two proteins in the extensor digitorum longus muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

No abstract

The levels of hsp27 and alpha B crystallin in various tissues of rats that had been subjected to heat stress were determined by specific immunoassays. When rats were immersed in a water bath at 42 degrees C for 20 min, the levels of hsp27 in most tissues, including central nervous tissue, liver, lung, spleen, adrenal glands, and hypophysis, had increased dramatically 8 to 16 h after the treatment. alpha B crystallin was also induced at high levels in the liver and adrenals, but not much was induced in the central nervous tissue. The increases in the levels of both hsp27 and alpha B crystallin in response to heat stress were markedly inhibited when ethanol or an alpha 1-adrenergic antagonist, prazosin, was administered before, but not after, the stress period. The expression of mRNA for hsp27 was suppressed in the livers of rats that had received ethanol or prazosin. A beta-adrenergic antagonist, propranolol, and an alpha 2-adrenergic antagonist, yohimbine, did not inhibit induction of the synthesis of the two proteins. Accumulation of hsp70 after heat stress, at least in the liver, was inhibited by ethanol or prazosin in a similar manner to that of hsp27, as detected on Western blot analysis. These results suggest that the induction of hsp27 and alpha B crystallin, as well as of hsp70, in tissues of heat-stressed rats is controlled by a physiological process(es) that is sensitive to ethanol and prazosin, and is operative for a short time during the application of heat stress.