Isolated gonadotropin-releasing hormone neurons harvested from adult male rats secrete biologically active neuropeptide in a regular repetitive manner.

Immunochemical treatments for the recovery of viable GnRH neurons from adult male rats have previously been described by this laboratory. In the present report, efforts were made to limit cellular adhesion, as well as the proteolytic and mechanical damage which occurred during isolation of the neurons, in order to determine if such damage may account for failure of the isolated cells to exhibit spontaneous neuropeptide release. These modifications prevented the loss of assayable GnRH during the isolation process, and neurons recovered from individual rats in this study contained 10.7 +/- 2.5 ng GnRH. Further, all isolated neuronal preparations exhibited spontaneous peptide release which continued in a regular repetitive manner. When maintained in closed chambers, these preparations released 105 +/- 42 pg/ml biologically active GnRH at 18.9 +/- 0.4-min intervals. In contrast, GnRH release from heterologous preparations was characterized by erratic low level pulses. The results from this work suggest that independent neuroendocrine properties of GnRH neurons may be responsible for tonic gonadotropin secretion in castrated adult male rats and that the erratic patterns of gonadotropin release in gonadally intact males may be related, in part, to coupling between GnRH neurons and unidentified neuronal factors.

Evidence suggesting that the two forms of heme oxygenase are products of different genes.

Recently, we have reported on the presence of two forms of heme oxygenase in rat liver and testis microsomes, referred to as HO-1 and HO-2 (M. D. Maines, G. M. Trakshel, and R. K. Kutty (1986) J. Biol. Chem. 261, 411-419; G. M. Trakshel, R. K. Kutty, and M. D. Maines (1986) J. Biol. Chem. 261, 11131-11137). Although the two forms differed in several biochemical properties, we could not ascertain whether they represented two isozymes or whether they were isoforms of heme oxygenase. In the present study, we provide evidence suggesting that the two forms are isozymes and represent different gene products. We also provide data suggesting that HO-1 is the commonly known heme oxygenase form. The molecular weight and immunochemical properties of HO-1 and HO-2 did not vary depending on the tissue source examined, i.e. liver and testis. Major differences, however, were noted in the amino acid composition of the two forms including the presence of 3 cysteine/cystine residues in HO-2 only. Using antibody to HO-2, four testis clones and two liver clones were isolated, and one liver and one testis clone were sequenced. Both clones revealed a 274-base-pair insert, and the sequence of both inserts was the same. The validity of assignment was confirmed by matching a 14-amino-acid peptide obtained from purified HO-2 with the sequence. Approximately 43% amino acid homology was detected between the HO-2 insert and the published amino acid sequence of heme oxygenase. However, amino acid homology search revealed the presence of two regions of homology: one 22-mer sequence with only one unmatched amino acid, and one 10-mer sequence with one unmatched amino acid. Heme oxygenase appeared to be the HO-1 form, an assignment based on its amino acid sequence matching the sequence of 2 peptides obtained from purified HO-1 and the immunochemical properties of the cobalt-, hematin-, and bromobenzene-induced rat liver enzyme. The secondary structure prediction analysis revealed an area of 100% structural homology with only 72% sequence homology. We predict this region may represent the catalytic site of the enzyme.