Differential expression of rat brain caspase family proteins during development and aging.

It is well recognized that caspases are essential effector molecules for carrying out apoptosis in eukaryotic cells. The expression of rat brain caspase family proteins (caspase-2, -3, -6, -7, -8, -9, 10) in development and aging was assessed using immunochemical detection. All of these caspases were expressed in the rat brain. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that cytosolic caspase-3, -7, -8, and -10 were highly expressed at E19, and decreased after birth. In contrast, cytosolic caspase-2, -6, and -9 were constitutively expressed from the early stages to 96 weeks of age. These results show that the expression of rat brain caspase family proteins is differentially regulated during the development and aging.     

Determining the pharmacokinetics of psilocin in rat plasma using ultra-performance liquid chromatography coupled with a photodiode array detector after orally administering an extract of Gymnopilus spectabilis

This study established ultra-performance liquid chromatography coupled with a photodiode array detector for determining psilocin and its pharmacokinetics in rat plasma after orally administering an extract of Gymnopilus spectabilis. The extract was separated on an ODS C18 column (2.3 μm, 100 mm × 2.1 mm I.D.) by gradient elution with (A) water containing 50mM AcONH(4) and (B) acetonitrile. The wavelength was set at 265 nm and the injection volume was 10 μL. Under these conditions, the calibration curve was linear over the concentration range 0.2-20 μg/mL with a correlation coefficient of r(2)=0.9992. The inter- and intraday precision levels were less than 7% and the accuracies (%) were within the range 92.0-102.5%. The method was sufficiently valid to be applied to a pharmacokinetics study of psilocin in rat plasma. The pharmacokinetic parameters of psilocin in rat plasma after the oral administration of a G. spectabilis extract were as follows: C(max), 0.43 ± 0.12 μg/mL; T(max), 90 ± 2.1 min; AUC(0→t), 1238.3 ± 96.4 (μg/mL) min; and T(1/2), 117.3 ± 40.3 min.     

Neuropeptide γ-(l-9)-Peptide: A Major Product of the Posttranslational Processing of γ-Preprotachykinin in Rat Tissues

Abstract: γ-Preprotachykinin mRNA is the most abundant tachykinin mRNA in rat tissues, but the pathway of posttranslational processing of its translation product is unknown. An antiserum was raised against the synthetic peptide Asp-Ala-Gly-His-Gly-Gln-lle-Ser-His [neuropeptide γ-(1-9)-peptide, equivalent to γ-preprotachykinin-(72-80)-peptide], that showed <1% reactivity with intact neuropeptide γ and other tachykinins. Neuropeptide γ-(1-9)-peptide was detected by radioimmunoassay in relatively high concentrations in extracts of regions of rat brain and gastrointestinal tract. These concentrations correlated with (r = 0.99), but were significantly (p < 0.05) less than, the concentrations of neurokinin A-like immunoreactivity. The neuropeptide γ-(1-9)-like immunoreactivity in an extract of rat brain was eluted from a reverse-phase HPLC column in a single fraction with the same retention time as synthetic neuropeptide γ-(1 -9)-peptide. The synthetic peptide did not contract or relax isolated rat trachea, superior mesenteric artery, stomach fundus, or ileum, and the peptide did not affect the ability of neuropeptide 7 to contract the rat fundus. It is concluded that, in rat tissues, Lys⁷⁰-Arg⁷¹ in 7-preprotachykinin is a major site of posttranslational processing, but the resulting product, neuropeptide γ-(1-9)-peptide, is neither an agonist nor an antagonist at the neurokinin-2 (NK-2) receptor.     

Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems

Specific high-affinity binding sites for 125I-alpha-bungarotoxin and (-)-[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I-alpha-bungarotoxin had similar Kd values of 1.5 x 10(-9) and 0.8 x 10(-9) M for rat and locust preparations, respectively; the corresponding values for the (-)-[3H]nicotine-binding site were 9.3 x 10(-9) and 1.7 x 10(-7) M. Methyllycaconitine (MLA) potently inhibited 125I-alpha-bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (-)-[3H]nicotine binding whereas (+)-anatoxin-a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)-anatoxin-a is a useful probe for the high-affinity nicotine-binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds alpha-bungarotoxin.     

A comparative study of neurotensin inactivation by brain peptidases from different vertebrate species

The products formed from mammalian neurotensin by peptidases in two subcellular fractions from rat, mouse, dove, terrapin and goldfish brain were separated and identified using high-performance liquid chromatography. The main neurotensin metabolites were [1-8]-, [1-10]- and [1-7]-sequences; goldfish and terrapin brain fractions also produced [1-11]- and [1-12]-fragments. Avian neurotensin was cleaved by peptidases in rat and dove brain fractions to [1-8]-, [9-13]-, [1-10]- and [1-12]-fragments. Similar mechanisms of inactivation were found for both mammalian and avian neurotensins .     

Multiple forms of choline acetyltransferase in several species demonstrated by isoelectric focusing

1. The behaviour of choline acetyltransferase from pigeon, guinea-pig, rat and cat brain on isoelectric focusing was studied. 2. Choline acetyltransferase from pigeon and guinea-pig brain showed single peaks with isoelectric points at pH6.6 and 6.8 respectively. Only one molecular form of the enzyme was therefore detected in these species. 3. Three peaks of choline acetyltransferase activities with isoelectric points 7.3-7.6, 7.7-7.9 and 8.3 were obtained with enzyme preparations from rat brain. 4. The separate identities of each of the three forms were confirmed by refocusing. 5. Choline acetyltransferase activity from a high-speed supernatant of rat brain homogenate was distributed similarly to a partially purified enzyme preparation from rat brain in the isoelectric gradient. 6. The enzyme activities from cat brain were separated into two distinct peaks with isoelectric points 7.0 and 8.4, and a possible third peak with isoelectric point 7.6. 7. The two main peaks showed considerable differences in stability on storage, and their identities were confirmed by refocusing. 8. The distribution of the enzyme activities was unaltered by isoelectric focusing in the presence of 3m-urea. 9. The apparent K(m) for choline of choline acetyltransferase from rat, cat and guinea-pig brain was 0.8mm, whereas for the pigeon enzyme it was 0.4mm.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Antiplasmodial activity of sesquilignans and sesquineolignans from Bonamia spectabilis

Phytochemical re-investigation of the aerial parts of Bonamia spectabilis (Convolvulaceae) led to the isolation of four minor tetrahydrofuran-type sesquilignans (bonaspectins E-H) together with the known neolignan virolongin A and the known lignan rel-(7S,8R,7'R,8'R)-3,3',4,4',5,5'-hexamethoxylignan. Their structures were established on the basis of spectral data. These six compounds as well as further seven lignanoids from B. spectabilis, characterised previously, were tested for their antiplasmodial activity against a chloroquine-sensitive strain (PoW) and a chloroquine-resistant clone (Dd2) of Plasmodium falciparum. Bonaspectin C 4"-O-glucoside, its aglycone, and bonaspectin D 4"-O-glucoside revealed the highest antiplasmodial activities (IC50 values: 1.3, 2.0, 6.5 microM [PoW]; 1.7, 4.6, 3.7 microM [Dd2], respectively).     

Acetoacetate metabolism in infant and adult rat brain in vitro

1. Acetoacetate or dl-beta-hydroxybutyrate increases the rate of oxygen consumption to a smaller extent than that brought about by glucose or pyruvate in adult rat brain-cortex slices but to the same extent as that in infant rat brain-cortex slices. 2. The rate of (14)CO(2) evolution from [1-(14)C]glucose considerably exceeds that from [6-(14)C]glucose in respiring infant rat brain-cortex slices, in contrast with adult brain-cortex slices, suggesting that the hexose monophosphate shunt operates at a greater rate in the infant rat brain than in the adult rat brain. 3. The rate of (14)CO(2) evolution from [3-(14)C]acetoacetate or dl-beta-hydroxy[3-(14)C]butyrate, in the absence of glucose, is the same in infant rat brain slices as in adult rat brain slices. It exceeds that from [2-(14)C]glucose in infant rat brain but is less than that from [2-(14)C]glucose in adult rat brain. 4. Acetoacetate is oxidized in the brain through the operation of the citric acid cycle, as shown by the accelerating effect of glucose on acetoacetate oxidation in adult brain slices, by the inhibitory effects of malonate in both infant and adult brain slices and by its conversion into glutamate and related amino acids in both tissues. 5. Acetoacetate does not affect glucose utilization in adult or infant brain slices. It inhibits the rate of (14)CO(2) formation from [2-(14)C]glucose or [U-(14)C]-glucose the effect not being wholly due to isotopic dilution. 6. Acetoacetate inhibits non-competitively the oxidation of [1-(14)C]pyruvate, the effect being attributed to competition between acetyl-CoA and CoA for the pyruvate-oxidation system. 7. Acetoacetate increases the rate of aerobic formation of lactate from glucose with both adult and infant rat brain slices. 8. The presence of 0.1mm-2,4-dinitrophenol diminishes but does not abolish the rate of (14)CO(2) formation from [3-(14)C]acetoacetate in rat brain slices. This points to the participation of ATP in the process of oxidation of acetoacetate in infant or adult rat brain. 9. The presence of 5mm-d-glutamate inhibits the rate of (14)CO(2) formation from [3-(14)C]acetoacetate, in the presence or absence of glucose. 10. Labelled amino acids are formed from [3-(14)C]acetoacetate in both adult and infant rat brain-cortex slices, but the amounts are smaller than those found with [2-(14)C]glucose in adult rat brain and greater than those found with [2-(14)C]glucose in infant rat brain. 11. Acetoacetate is not as effective as glucose as a precursor of acetylcholine in adult rat brain but is as effective as glucose in infant rat brain slices. 12. Acetoacetate or beta-hydroxybutyrate is a more potent source of acetyl-CoA than is glucose in infant rat brain slices but is less so in adult rat brain slices.     

Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain

The aims of this study were to determine the cellular and subcellular localization of aquaporin-9 (AQP9) in different rat organs by immunoblotting, immunohistochemistry and immunoelectron microscopy. To analyze this, we used rabbit antibodies to rat AQP9 raised against three different AQP9 peptides (amino acids 267-287, 274-295, and 278-295). In Cos7 cells transfected with rat AQP9, the affinity-purified antibodies exhibited marked labeling, whereas nontransfected cells and cells transfected with aquaporin-8 (AQP8) exhibited no labeling, indicating the specificity of the AQP9 antibodies. Immunoblotting revealed a predominant band of 28 kDa in membranes of total rat liver, epididymis, testes, spleen, and brain. Preabsorption with the immunizing peptides eliminated the labeling. Immunohistochemistry showed strong anti-AQP9 labeling in liver hepatocytes. The labeling was strongest at the sinusoidal surface, and there was little intracellular labeling. Immunoelectron microscopy revealed that the labeling was associated with the plasma membrane of the hepatocytes. In testes Leydig cells exhibited anti-AQP9 labeling, and in epididymis, the stereocilia of the ciliated cells (principal cells) exhibited significant labeling, whereas there was no labeling of the nonciliated cells (basal cells). This was confirmed by immunoelectron microscopy. In spleen strong labeling of cells was observed of leukocytes in the red pulp, whereas there was no labeling of cells in the white pulp. In rat brain, AQP9 immunolabeling was confined to ependymal cells lining the ventricles and to the tanycytes of the mediobasal hypothalamus. Antibody preabsorbed with the immunizing peptide revealed no labeling. In conclusion, AQP9 proteins is strongly expressed in rat liver, testes, epididymis, spleen, and brain.     

Neurochemical effects of the synthetic ACTH4-9-analog Hoe 427 (Ebiratide) in rat brain

The ACTH4-9-analog Hoe 427 systemically injected in a dose range from 0.01-10 micrograms/kg caused a fall in acetylcholine (ACh) content in different brain areas of the rat. This effect occurred 0.5 hour after a single administration and lasted up to 24 hours. The decrease in ACh content induced by Hoe 427 was more pronounced when the animals were pretreated with dexamethasone (over 7 days 1 mg/kg SC, daily). Coadministration of the choline uptake inhibitor hemicholinium-3 (HC-3) and Hoe 427 potentiated the decrease in ACh content induced by HC-3. In the same dose range Hoe 427 acutely evoked an increase of the activity of the enzyme choline acetyltransferase as well as an elevation of brain cyclic GMP content. These data indicate that Hoe 427 enhances ACh metabolism in rat brain after systemic administration.     

Bonaspectins and neobonaspectins, first sesquilignans and sesquineolignans from a convolvulaceous species

Four new tetrahydrofuran-type sesquilignans, named bonaspectin A, bonaspectin B, bonaspectin C 4'-beta-glucoside and bonaspectin D 4'-beta-glucoside, as well as two new 8.O.4'-type sesquineolignans, named neobonaspectin A and B, were isolated from the aerial vegetative parts of Bonamia spectabilis (Convolvulaceae), together with the known compound rel-(7S,8S,7'R,8'R)-3,3',4,4',5,5'-hexamethoxy-7.O.7',8.8'-lignan. Their structures were established on the basis of spectral data.     

Potential of Leguminous Cover Crops in Management of a Mixed Population of Root-knot Nematodes (Meloidogyne spp.)

Root-knot nematode is an important pest in agricultural production worldwide. Crop rotation is the only management strategy in some production systems, especially for resource poor farmers in developing countries. A series of experiments was conducted in the laboratory with several leguminous cover crops to investigate their potential for managing a mixture of root-knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica). The root-knot nematode mixture failed to multiply on Mucuna pruriens and Crotalaria spectabilis but on Dolichos lablab the population increased more than 2- fold when inoculated with 500 and 1,000 nematodes per plant. There was no root-galling on M. pruriens and C. spectabilis but the gall rating was noted on D. lablab. Greater mortality of juvenile root-knot nematodes occurred when exposed to eluants of roots and leaves of leguminous crops than those of tomato; 48.7% of juveniles died after 72 h exposure to root eluant of C. spectabilis. The leaf eluant of D. lablab was toxic to nematodes but the root eluant was not. Thus, different parts of a botanical contain different active ingredients or different concentrations of the same active ingredient. The numbers of root-knot nematode eggs that hatched in root exudates of M. pruriens and C. spectabilis were significantly lower (20% and 26%) than in distilled water, tomato and P. vulgaris root exudates (83%, 72% and 89%) respectively. Tomato lacks nematotoxic compounds found in M. pruriens and C. spectabilis. Three months after inoculating plants with 1,000 root-knot nematode juveniles the populations in pots with M. pruriens, C. spectabilis and C. retusa had been reduced by approximately 79%, 85% and 86% respectively; compared with an increase of 262% nematodes in pots with Phaseolus vulgaris. There was significant reduction of 90% nematodes in fallow pots with no growing plant. The results from this study demonstrate that some leguminous species contain compounds that either kill root-knot nematodes or interfere with hatching and affect their capacity to invade and develop within their roots. M. pruriens, C. spectabilis and C. retusa could be used with effect to decrease a mixed field populations of root-knot nematodes.     

Rat peptidylglycine alpha-amidating enzyme: the relation between activities at neutral and alkaline pH Values

A substantially high level of alpha-amidating activity at an alkaline pH (8-9.5), often seen as another pH optimum peak in addition to the neutral one, has been observed in various rat tissues. We have also found that crude enzymes from rat brain, pituitary, and small intestine showed a pH profile with two pH optima at neutral pH (6.5-7) and alkaline pH (8.5-9) when D-Tyr-Val-Gly was used as substrate. With a combination of ion-exchange and gel filtration chromatographies, we obtained two fractions, S-1 and S-2, from rat brain; S-1 contained an alpha-amidating enzyme of an apparent molecular weight of 36,000 (36K enzyme) exhibiting a single pH optimum at 8.5. On the other hand, S-2 apparently showed almost no or only marginal activity at either pH 7 or 8.5, but when S-2 was combined with S-1, a neutral pH optimum at 7 could be elicited. The factor in S-2 that was responsible for this combined action was a protein of an apparent molecular weight of 41,000 (41K protein). Both proteins were found to be colocalized in the same subcellular organelle, probably in the secretory granule. It seems likely, then, that the pH profiles characterized by two optimal peaks seen in crude rat enzymes can be attributed to the presence at an appropriate ratio of the 41K protein and 36K enzyme.     

Cryopreservation of organotypic brain spheroid cultures

The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5-15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12-15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5-7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP: phosphatidate cytidylyltransferase and CDPdiacylglycerol: inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7–10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP: phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Investigation of PAR-1-type thrombin receptors in rat glioma C6 cells with a novel monoclonal anti-PAR-1 antibody (Mab COR7-6H9)

Rat glioma C6 cells have been demonstrated to be a suitable model in the investigation of PAR-1-type thrombin receptors in brain. However, anti-PAR-1 antibodies, which should be very helpful tools in studying PAR-1 in rat cells, have not been available up until now. Therefore, we prepared a monoclonal anti-thrombin receptor antibody (Mab COR7-6H9) directed against the peptide sequence GRAVYLNKSRFPPMPPPPFISEDASG in the N-terminus below the thrombin cleavage site of the rat PAR-1-type thrombin receptor. Using this antibody, we demonstrated the presence of PAR-1 binding sites on the plasma membrane of rat glioma C6 cells both with confocal laser fluorescence and with scanning electron microscopy. In addition, Mab COR7-6H9 was shown to block PAR-1-mediated transmembranal signaling as demonstrated by measurement of free intracellular calcium and cyclic AMP. This novel anti-PAR-1 antibody is therefore likely to be a very helpful tool in studying PAR-1-type thrombin receptors in rat brain.     

Enantioselective hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction

Rates of hydrolysis of racemic and enantiomeric oxazepam 3-acetates (OXA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. When rac-OXA was the substrate, esterases in human and rat liver microsomes were highly enantioselective toward (R)-OXA. In contrast, esterases in rat brain S9 fraction were highly enantioselective toward (S)-OXA. Hydrolysis rates of rac-OXA were highly dependent on the amount of esterases used. At 0.05 mg protein equivalent of esterases and 150 nmol of rac-OXA per ml of incubation mixture, the (R)-OXA was hydrolyzed 3.6-fold and 18.5-fold faster than (S)-OXA by rat and human liver microsomes, respectively. The specific activities (nmol of OXA hydrolyzed/mg microsomal protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-OXA were approximately 120 (rat) and 1,980 (human), and in the hydrolysis of enantiomerically pure (S)-OXA were 4 (rat) and 7 (human), respectively. In the incubation of rac-OXA with rat brain S9 fraction, (S)-OXA was hydrolyzed approximately 6-fold faster than (R)-OXA. Results also indicated an enantiomeric interaction in the hydrolysis of rac-OXA by esterases in rat and human liver microsomes; the presence of (R)-OXA stimulated the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA inhibited the hydrolysis of (R)-OXA. In rat brain S9 fraction, the presence of (R)-OXA inhibited the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA appeared to have stimulated the hydrolysis of (R)-OXA.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.