Archivesof Oto-Rhino-Laryngology 9 Springer-Verlag 1978
Arch. Otorhinolaryngol.221, 311-312 (1978)
Reevaluation of Adenylate Cyelase in Reissner's Membrane* I. Thalmann and R. Thalmann Department of Otolaryngology,Washington University Medical School, 517 S. Euclid, St. Louis, MO 63110, USA We have previously reported that the activity of adenylate cyclase of Reissner's membrane was less than 0.5 pmol/mg dry weight/min (Paloheimo and Thalmann, 1977). This activity is at least four times lower than the activity in the spiral ganglion and 30 and 40 times lower than the activity in organ of Corti and stria vascularis, respectively. During the course of subsequent studies concerning the influence of stimulators of adenylate cyclase, we found that the low enzyme values in Reissner's membrane were due to an artifact caused by unspecific binding. The original analytical method as described by Paloheimo and Thalmann (1977) was, therefore, modified in the following way" After incubation the reaction was stopped instead of by heating, by the addition of 7.5% trichloroacetic acid. The trichloroacetic acid was removed by four washings with water-saturated ether and subsequent biodrying, thereby eliminating any possibility of unspecific binding. The activity of Reissner's membrane determined with this improved procedure was found to be 7.4 + 1.3 (S.D.) pmol/mg dry weight/min in seven determinations. 5 m M NaF increased the activity to 32.5 + 1.3 (7) pmol/mg dry weight/min which represents a characteristic response of the enzyme to this stimulant. The enzyme activities in organ of Corti and stria vascularis were found to be in the same range as previously reported. We have not yet identified the reason why the artifact due to unspecific binding occurred only in Reissner's membrane; one possibility may be the high surface to volume ratio of this delicate tissue sheet. The presence of a relatively high activity of adenylate cyclase is in line with a rather high steady state level of cyclic AMP in Reissner's membrane (3.89 pmol/mg dry weight; Thalmann et al., 1979). This value is only slightly lower than that of organ of Corti and stria vascularis. Ross (1978) demonstrated a strong increase of glycogen deposits in Reissner's membrane following chemical sympathectomy with 6-hydroxydopamine. This investigator proposed that this effect is due to the absence of catecholamines which
*
Supported by NIH grant 06575, and NSF grant BNS 77-16842 R. Thalmann, M.D. (address see above)
Offprint requests to:
0302-9530/78/0221/0311/$ 01.00
312
I. Thalmann and R. Thalmann
normally stimulate adenylate cyclase which in turn activates glycogen phosphorylase and consequent glycogenolysis. Our revised results indicating a substantial activity o f adenylate eyclase in Reissner's membrane, are compatible with Ross' conclusions.
References Paloheimo, S., Thalmann, R.: Influence of "loop" diuretics upon Na+K+-ATPase and adenylate cyclase of the stria vascularis, Arch. Otorhinolaryngol. (N.Y.) 217, 347-359 (1977) Ross, M. D.: Glycogen accumulation in Reissner's membrane following chemical sympathectomy with 6-hydroxydopamine. Acta Otolaryngol. (Stockh.) (in press) (1978) Thalmann, R., Paloheimo, S., Thalmann, I.: Distribution of cyclic AMP and cyclic GMP in the cochlea. Acta Otolaryngol. (Stoekh.) (submitted) (1979) Received September 20, 1979
Arch. Otorhinolaryngol.221, 311-312 (1978)
Reevaluation of Adenylate Cyelase in Reissner's Membrane* I. Thalmann and R. Thalmann Department of Otolaryngology,Washington University Medical School, 517 S. Euclid, St. Louis, MO 63110, USA We have previously reported that the activity of adenylate cyclase of Reissner's membrane was less than 0.5 pmol/mg dry weight/min (Paloheimo and Thalmann, 1977). This activity is at least four times lower than the activity in the spiral ganglion and 30 and 40 times lower than the activity in organ of Corti and stria vascularis, respectively. During the course of subsequent studies concerning the influence of stimulators of adenylate cyclase, we found that the low enzyme values in Reissner's membrane were due to an artifact caused by unspecific binding. The original analytical method as described by Paloheimo and Thalmann (1977) was, therefore, modified in the following way" After incubation the reaction was stopped instead of by heating, by the addition of 7.5% trichloroacetic acid. The trichloroacetic acid was removed by four washings with water-saturated ether and subsequent biodrying, thereby eliminating any possibility of unspecific binding. The activity of Reissner's membrane determined with this improved procedure was found to be 7.4 + 1.3 (S.D.) pmol/mg dry weight/min in seven determinations. 5 m M NaF increased the activity to 32.5 + 1.3 (7) pmol/mg dry weight/min which represents a characteristic response of the enzyme to this stimulant. The enzyme activities in organ of Corti and stria vascularis were found to be in the same range as previously reported. We have not yet identified the reason why the artifact due to unspecific binding occurred only in Reissner's membrane; one possibility may be the high surface to volume ratio of this delicate tissue sheet. The presence of a relatively high activity of adenylate cyclase is in line with a rather high steady state level of cyclic AMP in Reissner's membrane (3.89 pmol/mg dry weight; Thalmann et al., 1979). This value is only slightly lower than that of organ of Corti and stria vascularis. Ross (1978) demonstrated a strong increase of glycogen deposits in Reissner's membrane following chemical sympathectomy with 6-hydroxydopamine. This investigator proposed that this effect is due to the absence of catecholamines which
*
Supported by NIH grant 06575, and NSF grant BNS 77-16842 R. Thalmann, M.D. (address see above)
Offprint requests to:
0302-9530/78/0221/0311/$ 01.00
312
I. Thalmann and R. Thalmann
normally stimulate adenylate cyclase which in turn activates glycogen phosphorylase and consequent glycogenolysis. Our revised results indicating a substantial activity o f adenylate eyclase in Reissner's membrane, are compatible with Ross' conclusions.
References Paloheimo, S., Thalmann, R.: Influence of "loop" diuretics upon Na+K+-ATPase and adenylate cyclase of the stria vascularis, Arch. Otorhinolaryngol. (N.Y.) 217, 347-359 (1977) Ross, M. D.: Glycogen accumulation in Reissner's membrane following chemical sympathectomy with 6-hydroxydopamine. Acta Otolaryngol. (Stockh.) (in press) (1978) Thalmann, R., Paloheimo, S., Thalmann, I.: Distribution of cyclic AMP and cyclic GMP in the cochlea. Acta Otolaryngol. (Stoekh.) (submitted) (1979) Received September 20, 1979
