Proteolytic resistance and biological activity of N-methylated analogs of [pGlu6] substance P6-11.

Five synthetic N-methylated analogs (II-V) of the C-terminal hexapeptide analog of substance P (SP), [pGlu6]SP6-11 (I) were evaluated for their metabolic stability and in vitro spasmogenic activity. The metabolic resistance of the analogs was tested by two SP degrading systems with different specificities, namely, the rat parotid and the hypothalamic slice systems. Their biological activity was assessed in the isolated guinea pig ileum. The analog [pGlu6, N-Me Phe7, N-Me Gly9]SP6-11 (III), had relative potency of 65% in the spasmogenic assay as compared to the parent compound. It was found to be more stable than the parent peptide in the hypothalamus, whereas in the parotid system it was susceptible as the parent peptide. However, the analog [pGlu6, N-Me Leu10]SP6-11 (II) (46% relative potency in the spasmogenic assay) was more stable than the parent peptide in the parotid system but did not show any improved stability in the hypothalamus. Identification of degradation products of the [pGlu6, N-Me Leu10]SP6-11 reflected the differences in the specificities of the two preparations. A significant drop in potency (7%) was observed for [pGlu, N-Me Phe7]SP6-11 (IV). This analog was more stable in the hypothalamic system than in the parotid. Introduction of a double methylation, [pGlu6, N-Me Leu10] SP6-11, contributed toward the stabilization in both degrading systems. Its relative spasmogenic activity was comparable to that of analog IV. In light of the above mentioned findings the implications of the N-methylated analogs with respect to putative CNS activity are discussed.

Coupling of photoexcited rhodopsin to inositol phospholipid hydrolysis in fly photoreceptors.

Fly photoreceptor membranes were used to test the effect on defined biochemical reactions of light and of compounds causing photoreceptor excitation. Complementary electrophysiological studies examined whether putative second messengers excite the fly photoreceptor cells. This analysis revealed the following sequence of events: photoexcited rhodopsin activates a G protein by facilitating GTP binding. The G protein then activates a phospholipase C that generates inositol trisphosphate, which in turn acts as an internal messenger to bring about depolarization of the photoreceptor cell. Binding assays of GTP analogs and measurements of GTPase activity showed that there are 1.6 million copies of G protein per photoreceptor cell. The GTP binding component is a 41-kDa protein, and the light-activated GTPase is dependent on photoconversion of rhodopsin to metarhodopsin. Analysis of phospholipase C activity revealed that this enzyme is under stringent control of the G protein, that the major product formed is inositol trisphosphate, and that this product is rapidly hydrolyzed by a specific phosphomonoesterase. Introduction of inositol trisphosphate to the intact photoreceptor cell mimics the effect of light, and bisphosphoglycerate, which inhibits inositol trisphosphate hydrolysis, enhances the effects of inositol trisphosphate and of dim light. The interaction of photoexcited rhodopsin with a G protein is thus similar in both vertebrate and invertebrate photoreceptors. These G proteins, however, activate different photoreceptor enzymes: phospholipase C in invertebrates and cGMP phosphodiesterase in vertebrates.

Metabolically stable analogues of substance P: persistent action of partially modified retro-inverso analogues of substance P on rat parotid and hypothalamic slices.

In a search for metabolically stable analogues of substance P (SP) the hexapeptide [pGlu6]SP-(6-11) was modified by reversal of the direction of a single amide bond. This novel peptide modification reverses the direction of the amide bonds at the peptide backbone but attempt to retain the topology of the amino acid side-chains at the peptide surface. The partial retro-inverso modification was successfully applied in a previous study for enkephalin analogues which were found to have potent and protracted morphinomimetic activity both in vivo and in vitro. The partially modified retro-inverso analogues: [pGlu6 psi(NH-CO)(RS)-Phe7]SP-(6-11) (analogue II) and [pGlu6,Phe8 psi(NH-CO)Gly9]SP-(6-11) (analogue III) were tested on guinea-pig ileum and for K+ release from rat parotid slices. Metabolic stability of the analogues was measured by their ability to produce persistent K+ release from parotid slices, their half life time (t1/2) in the rat parotid and hypothalamic slice systems and their resistance to proteolytic cleavage by chymotrypsin, pepsin, papain and pronase. Analogue II was devoid of biological activity and was slowly degraded in the parotid system and by several proteases. Analogue II was a full agonist of the SP-P receptor with a potency of 22 and 15% of the parent compound I, in the guinea-pig ileum and parotid slice system respectively. Pretreatment of the guinea-pig ileum with atropine (0.3 microM) had no effect on the potency of analogue III. On the other hand, when tested on rat vas deferens (an SP-E system), analogue III was about 20-fold more potent than the parent compound I.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P degrading systems of rat parotid and hypothalamus.

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu6]substance P6-11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K+ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu6]substance P6-11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe8-Gly9 with minor cleavage sites between Phe7-Phe8 and Gly9-Leu10. In the rat parotid slice system the major cleavage occurs between Gly9-Leu10 with a minor cleavage site between Phe7-Phe8. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH2, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.