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The main somatostatin-degrading proteases were purified from rat and pig brain homogenates and characterized as thiol-and metal-dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68-kDa protease cleaved somatostatin at three bonds (Asn5-Phe6, Phe6-Phe7 and ThrlO-Phell) and neurotensin only at the Arg8-Arg9 bond, whereas the 70-kDa protease digested somatostatin at only two bonds (Phe6-Phe7 and ThrlO-Phel 1) and neurotensin as well as acetylneurotensin-(8 -13) additionally (pig protease) or almost exclusively (rat protease) at the ProlOTyrll bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin 11, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68-kDa protease was found to be mainly, but not exclusively, soluble and not membraneassociated, whereas the inverse was detected for the 70-kDa protease. Based on distinct molecular and catalytic properties, the 68-kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70-kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensindegrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites. but with species-dependent activity. Species-independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin-(8 -13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.The cyclic tetradecapeptide somatostatin, originally purified as an inhibitory factor for the release of somatotropin from pituitary cells [l], is a widespread neurotransmitter or neuromodulator in the brain [2]. Somatostatin Enzymes. Soluble mctallo-endopeptidase (EC 3.4.24.1 5); neurotensin-degrading neutral metallopeptidase (EC 3.4.24.16); endo-oligopeptidase (EC 3.4.22.19); tissue-endopeptidase degrading collagenase-synthetic substrate, Pz-peptidase (EC 3.4.99.31); enkephalinase, membrane metallo-endopeptidase (EC 3.4.24.1 1); prolyl endopeptidase (thiol-dependent) (EC 3.4.22.18); cytosol aminopeptidase, leucyl aminopeptidase (EC 3.4.1 1.1); aminopeptidase M or N, microsomal (alanine) aminopeptidase (EC 3.4.1 1.2). vated to avoid a constant stimulus. However, as for other neuropeptides, the factors responsible for inactivation in the brain are insufficiently known [8 -lo].Recently, we have shown that somatostatin is inactivated by proteolytic degradation after exposure to cultivated neuronal and glial cells from rat brain [ll]. Hydrolysis...

Several neuropeptides, including neurotensin, somatostatin, bradykinin, angiotensin II, substance P, and luteinizing hormone-releasing hormone but not vasopressin and oxytocin, were actively metabolized through proteolytic degradation by cultivated astrocytes obtained from rat cerebral cortex. Because phenanthroline was an effective degradation inhibitor, metalloproteases were responsible for neuropeptide fragmentation. Neurotensin was cleaved by astrocytes at the Proio-Tyr" and Arg8-Argg bonds, whereas somatostatin was cleaved at the Phe6-Phe' and Thrio-Phe" bonds. These cleavage sites have been found previously with endopeptidases 24.1 6 and 24.1 5 purified from rat brain. Addition of specific inhibitors of these proteases, the dipeptide Pro-lle and N-[1-(RS )-carboxy-3-phenylpropyl]-Ala-Ala-Phe-4-aminobenzoate, significantly reduced the generation of the above neuropeptide fragments by astrocytes. The presence of endopeptidases 24.1 6 and 24.1 5 in homogenates of astrocytes could also be demonstrated by chromatographic separations of supernatant solubilized cell preparations. Proteolytic activity for neurotensin eluted after both gel and hydroxyapatite chromatography at the same positions as found for purified endopeptidase 24.16 or 24.15. In incubation experiments or in chromatographic separations no phosphoramidon-sensitive endopeptidase 24.1 1 (enkephalinase) or captopril-sensitive peptidyl dipeptidase A (angiotensin-converting enzyme) could be detected in cultivated astrocytes. Because astrocytes embrace the neuronal synapses where neuropeptides are released, we presume that the endopeptidases 24.1 6 and 24.15 on astrocytes are strategically located to contribute significantly to the inactivation of neurotensin, somatostatin, and other neuropeptides in the brain. Key Words: Neurotensin-Somatostatin-Bradykinin-Astrocytes-Endopeptidase 24.1 6-Endopeptidase 24.1 5-Degradation.

The recent availability of isotope-labelled somatostatin analogues has allowed one to detect somatostatin receptors in normal tissue as well as in endocrine or non-endocrine cranial tumours. The purpose of the present study was to establish the value of somatostatin receptor scintigraphy using an 111Indium-labelled somatostatin analogue, octreotide, in the diagnostic work-up of meningioma patients. Twenty-two patients (16 women, 6 men, aged from 19-70 years) with newly diagnosed, residual or recurrent cranial meningiomas were examined. 111Indium-labelled DTPA-octreotide was injected i.v.. Planar and tomographic images were obtained with a gamma camera 4-6, and 24 hours after injection. In all of the meningiomas studied a high density of somatostatin receptors was detected by scintigraphy. No false negative test result was found. Due to this, a 100% predictive value of a negative test was calculated. However, when the tumours were taken in culture differing staining intensity could be seen in the light- and electron microscopic level even on individual cells of a single culture when silver intensified somatostatin-gold was used as ligand. We conclude, that in vivo somatostatin receptor scintigraphy may aid in the pre-operative differential diagnosis of skull base tumours.

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