Regulation of pp60(c-src) synthesis in rat hippocampal slices by in vitro ischemia and glucocorticoid administration.

Corticosteroids, released from the adrenal gland in response to stress, bind to receptors that act as transcription factors to alter gene expression and, subsequently, protein synthesis. Using [(35)S]-methionine-cysteine incorporation to measure protein synthesis in hippocampal slices incubated under ischemic conditions, synthesis of 60 kDa and 78 kDa proteins decreases 4 hr after in vivo administration of corticosterone to rats. The former protein has been identified by immunoblotting and immunoprecipitation to be the proto-oncogene, pp60(c-src). In the absence of prior glucocorticoid administration, ischemic conditions increase the amount of immunoreactive pp60(c-src) in membranes of hippocampal slices. Chronic exposure to elevated titers of glucocorticoids has been demonstrated to result in cell loss as well as in reduced neuronal plasticity and regeneration. Given the involvement of pp60(c-src) in synaptic plasticity and cell growth, glucocorticoid-mediated reduction in its synthesis is a potential molecular marker for stress-induced alterations in brain function.

Glucocorticoids regulate the synthesis of HSP27 in rat brain slices.

Using mild heat shock of rat brain slices as a model for cellular insult, corticosteroid-mediated regulation of protein synthesis has been investigated. Following a single in vivo injection of rats with corticosterone or the Type II glucocorticoid receptor agonist, RU-28362, synthesis of a 28 kDa protein is elevated in cerebellar slices which are subsequently incubated in vitro at 39 degrees C for 3 h. Immunoblotting of proteins subsequent to separation by two-dimensional gel electrophoresis has identified this glucocorticoid-sensitive protein to be the small molecular weight heat-shock protein, HSP27. Synthesis of the major heat-shock proteins, HSP70 and HSP90, is not glucocorticoid-sensitive. When animals are sacrificed at either 4 h following an aldosterone injection or at 24 h following a corticosterone injection, the synthesis of HSP27 in cerebellar slices is decreased. Treatment of adrenalectomized rats with either corticosterone, RU-28362 or aldosterone produces increased synthesis of HSP27. With duration of heat shock, there is a transient increase in the synthesis of HSP27 after 2 h at 39 degrees C in slices from the cerebral cortex, with a more sustained synthesis of HSP27 in cerebellar slices. In hippocampal slices, HSP27 is rarely present. The upregulated synthesis of HSP27 in the cerebellum following an acute exposure to stress-like elevations in corticosterone titers may contribute to the relative resistance of this brain region to cellular insults.

Muscarinic receptor- and phorbol ester-stimulated phosphorylation of protein kinase C substrates in adult and neonatal cortical slices.

The neuron-specific protein B-50 (GAP-43) is a major presynaptic substrate for protein kinase C (PKC). Phosphorylation of B-50 by PKC at serine-41 is functionally related to signal transduction in association with process outgrowth and neurotransmitter release. Thus, it is important to characterize the factors which modulate phosphorylation of B-50 by PKC. Phosphoinositide (PI)-coupled muscarinic acetylcholine receptor (mAchR) activation would be expected to increase PKC activity through production of the second messenger, diacylglycerol. To test the hypothesis that activation of mAchR also increases phosphorylation of B-50, protein phosphorylation has been examined in cerebral cortical slices in response to the cholinergic agonist, carbachol (Cch) in comparison to the phorbol ester, 4beta-phorbol 12, 13-dibutyrate (PDB), a known activator of PKC. At short times of incubation with 1 mM Cch, a concentration which maximally activates PI metabolism, increased phosphorylation of a group of synaptosomal proteins, including B-50 and myristoylated, alanine-rich C kinase substrate (MARCKS), was observed. This increase was approximately half of that obtained in response to 1 microM PDB. Differing patterns of protein phosphorylation were observed in neonatal and adult slices: neonatal samples contained more MARCKS and a PKC substrate with a Mr of 46 kDa. Phosphorylation of B-50 and MARCKS was sensitive to Cch in both cases. Immunoblotting demonstrated less m1 acetylcholine receptor (the predominant mAchR subtype coupled to PI metabolism in the cortex) in neonatal, as compared to adult, synaptosomal fractions. These results are consistent with a coupling between mAchR-stimulated PI metabolism and PKC-mediated protein phosphorylation that is developmentally regulated.

Glucocorticoids regulate protein synthesis in hippocampal slices under mild heat shock conditions.

Glucocorticoid hormones potentiate the toxic effects of neuronal stressors. Alteration of gene expression by glucocorticoids could contribute to neuronal susceptibility by downregulating the synthesis of proteins necessary to adapt to challenge. Using heat shock of hippocampal slices as a model for cellular insult, protein synthesis has been examined in response to acute glucocorticoid administration to rats. Incubation of hippocampal slices at 39 degrees C produces a heat-shock pattern of protein synthesis in that total incorporation of labeled amino acid is diminished, whereas synthesis of the major heat-shock proteins, HSP90 and HSP70, is increased. Prior administration of corticosterone to rats does not affect subsequent synthesis of HSP90 or HSP70 in slices. However, at 4 or 24 h following a single corticosterone injection, the synthesis of two acidic proteins is found to be altered: a 25-kDa protein is downregulated in the nuclear and synaptosomal-mitochondrial fraction of the hippocampus, and a 47-kDa protein is downregulated in all three fractions of the hippocampus, cortex, and cerebellum. These effects are mimicked by administration of RU-28362, a specific glucocorticoid (GR or Type II) receptor agonist. Since decreased synthesis of p25 and p47 is the only glucocorticoid-mediated response observed in slices under heat-shock conditions, these proteins may be related to the adaptation to heat shock.

Regulation of in vitro phosphorylation of the casein kinase II sites in B-50 (GAP-43).

Casein kinase II (CKII) phosphorylates the rat neuronal growth-associated protein B-50 (GAP-43) at serines 191/192 and threonines 88, 89 and/or 95 both in vitro and in neuronal growth cones. Since little is known concerning regulation of the phosphorylation of these sites, these studies were undertaken to characterize the factors which determine the degree of B-50 phosphorylation by CKII in vitro. Phosphorylation of rat B-50 on serine and threonine residues by recombinant human CKII is stimulated by polylysine. Maximal stimulation occurs at 10 microg/ml of polylysine, a concentration which has no effect on protein kinase C (PKC)-mediated phosphorylation of B-50. Digestion with Staphylococcus aureus V8 protease demonstrates CKII-mediated phosphorylation of B-501-132 and the C-terminal fragment S3/S4. Phosphorylation of B-50 by either CKII or PKC is inhibited by the N-terminal monoclonal antibody NM2, while the C-terminal antibody NM6 has no effect on phosphorylation by either protein kinase. Protein phosphatase 2A dephosphorylates both the CKII and PKC sites, while protein phosphatases 2B and 1 are more selective for the PKC site. These results indicate that the phosphorylations of B-50 by CKII and PKC are determined by distinct regulatory signals in vivo.

Variations on a theme in g.

G Proteins, Receptors, and Disease Edited by Allen M. Spiegel. Totowa, Humana, 1998, $135.00 (x+324 pages), ISBN 0-896-034305.

Adenosine 3',5'-cyclic monophosphate/vanadate-sensitive phosphorylation of DARPP-32- and inhibitor-1-immunoreactive proteins.

Among the cellular actions of vanadate ions are several that have the potential to be of significance in the regulation of protein phosphorylation. The effects of vanadate on adenosine 3',5' cyclic monophosphate (cAMP)-dependent and independent, alkali-resistant protein phosphorylation in a synaptosomal preparation from rat cortex were examined in this study. Three major vanadate-stimulated, cAMP-independent phosphoproteins (58-, 50-, and 39-kDa) and two cAMP-dependent species (37- and 32-kDa) were detectable. The potentiation between vanadate and cAMP in stimulating the phosphorylation of the latter two proteins is in contrast to the nonadditive combined effect of both on the phosphorylation of other synaptosomal proteins. The two cAMP-dependent, 32P-labeled proteins possess identical or very similar physicochemical properties to two previously cited neuronal phosphoproteins, namely, dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein-32 (DARPP-32) and inhibitor-1 (I-1). Such properties include phosphorylation by cAMP-dependent protein kinase, the presence of an alkali-resistant phosphothreonine residue, comigration on two-dimensional gel electrophoresis, dephosphorylation by type-2B protein phosphatase, and crossreactivity with specific antibodies. Costimulation by cAMP and vanadate of phosphorylation of the latter two proteins on threonine residues, at concentrations of vanadate consistent with the regulation of protein tyrosine phosphatase activity, indicates a unique interaction between these two regulators of protein phosphorylation at the nerve terminus.

Phosphorylation of the casein kinase II domain of B-50 (GAP-43) in rat cortical growth cones.

Growth-associated phosphoprotein B-50 is a neural protein kinase C (PKC) substrate enriched in nerve growth cones that has been implicated in growth cone plasticity. Here we investigated whether B-50 is a physiological substrate for casein kinase II (CKII) in purified rat cortical growth cone preparations. Using site-specific proteolysis and known modulators of PKC, in combination with immunoprecipitation, mass spectrometry, and phosphoamino acid analysis, we demonstrate that endogenous growth cone B-50 is phosphorylated at multiple sites, on both serine and threonine residues. Consistent with previous reports, stimulation of PKC activity increased the phosphorylation of only those proteolytic fragments containing Ser41. Under basal conditions, however, phosphorylation was predominantly associated with fragments not containing Ser41. Mass spectrometry of tryptic digests of B-50, which had been immunoprecipitated from untreated growth cones, revealed that in situ phosphorylation occurs within peptides B-50(181-198) and B-50(82-98). These peptides contain the major and minor in vitro CKII phosphosites, respectively. In addition, cyanogen bromide digestion of immunoprecipitated chick B-50 generated a 4-kDa C-terminal B-50 phosphopeptide, confirming that phosphorylation of the CKII domain occurs across evolutionary diverse species. We conclude that B-50 in growth cones is not only a substrate for PKC, but also for CKII.

Corticosteroid-induced proteins in brain.

Cumulative exposure of hippocampal neurons to stress-like levels of corticosterone produces a negative spectrum of cellular alterations from ultrastructural changes to disruption of dendritic morphology and eventual degeneration. An experimental system which adapts itself to characterization of corticosteroid-induced proteins which mediate such effects is the hippocampal slice incubated in the presence of a radiolabeled amino acid following treatment of rats with corticosterone. The most consistently observed response to elevated corticosterone levels produced by exogenous injection is synthesis of a hippocampal cytosolic protein which has characteristics of glycerol phosphate dehydrogenase. Because synthesis of this protein is enhanced with a short latency as serum corticosterone levels are increased and terminated quickly upon re-establishment of basal conditions, it serves as a valid biological marker of the response of the hippocampus to short-term stress. In contrast, alterations in synthesis of other proteins following corticosterone treatment only become apparent under defined conditions or after chronic treatment. For example, steroid-inhibited synthesis of a hippocampal protein with an approximate molecular weight of 25,000 is only observed when slices are incubated at an elevated temperature. Such negative changes may represent loss of adaptive responses that protect the neuron from damage by cellular insults.

Phorbol ester- and glutamate-sensitive phosphorylation of hippocampal membrane proteins from adult and neonatal rats.

The phosphoinositide (PI) second messenger system in the neonatal rat brain is differentially stimulated as compared to that of the adult by agonists such as glutamate. Among the factors that might contribute to the neonatal pattern is the nature of phosphorylated membrane-bound proteins which could regulate this receptor-mediated response. This study was undertaken to compare membrane protein phosphorylation under conditions that affect PI hydrolysis in neonatal and adult rat hippocampus. Two-dimensional gel analysis revealed enhanced basal phosphorylation of two membrane proteins (M(r): 46,000 and 80,000; pI: 4.4 and 4.2, respectively) in the neonatal hippocampus when compared to the adult. The former phosphoprotein is present only in neonatal hippocampus. Phosphorylation of a 48,000 M(r) protein with a pI of 4.5 is prominent in hippocampal membranes from both neonatal and adult rats. After incubation of neonatal hippocampal slices with an active phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), all three proteins show decreased post-hoc phosphorylation. Slices from neonatal rats incubated with glutamate demonstrated no alteration in the phosphorylation of any of these proteins, while those from adult rats produced a marked change in phosphorylation of the 80,000 M(r) protein. The data suggest that phosphorylation of this protein from neonates is not yet as efficiently coupled to receptor stimulation as that from the adult. Immunoblot analysis revealed that the 48,000 M(r) protein is the growth-associated protein B-50/GAP-43 and that the 80,000 M(r) protein is a membrane-associated form of the MARCKS protein.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the regulatory properties of striatal and cortical adenylate cyclase.

Biochemical properties of adenylate cyclase in striatal and cortical membranes have been analyzed in parallel with their regulation by cholinergic compounds. Striatal adenylate cyclase is more sensitive to forskolin, while the cortical enzyme is more stimulated by GTP. In the presence of GTP, more inhibition by acetylcholine is seen in the cortex than in the striatum. Acetylcholine inhibits striatal adenylate cyclase activity equally in the presence or absence of forskolin but has a diminished ability to inhibit forskolin-stimulated adenylate cyclase in the cortex. The greater sensitivity of cortical muscarinic receptor-coupled adenylate cyclase to EGTA and calcium indicates predominant involvement of the calcium/calmodulin-dependent subtype of the enzyme. The relative effectiveness of antagonists, demonstrating an order of potency of atropine > amitriptyline > pirenzepine > gallamine in reversing the inhibition of adenylate cyclase by acetylcholine for both brain regions, suggests predominantly m4 receptor-mediated responses. These results suggest an m4-type receptor may be coupled to subtypes of adenylate cyclase in the striatum and cortex which differ in their biochemical properties.

Protein phosphatases 1 and 2A dephosphorylate B-50 in presynaptic plasma membranes from rat brain.

The protein B-50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phosphatase type 1 and 2A (PP-1 and PP-2A)-like activities. The present studies further demonstrate that B-50 is dephosphorylated not only by a spontaneously active PP-1-like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 micrograms of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor-2 and by high concentrations (microM) of okadaic acid, identifying it as a latent form of PP-1. In the presence of inhibitor-2 to block PP-1, histone H1 (16-64 micrograms/ml) and spermine (2 mM) increased B-50 dephosphorylation. This sensitivity to polycations and the reversal of their effects on B-50 dephosphorylation by 2 nM okadaic acid are indicative of PP-2A-like activity. PP-1- and PP-2A-like activities from SPM were further displayed by using exogenous phosphorylase alpha and histone H1 as substrates. Both PP-1 and PP-2A in rat SPM were immunologically identified with monospecific antibodies against the C-termini of catalytic subunits of rabbit skeletal muscle PP-1 and PP-2A. Okadaic acid-induced alteration of B-50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity-purified anti-B-50 immunoglobulin G. These results provide further evidence that SPM-bound PP-1 and PP-2A-like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B-50.

Stereoselective binding and activity of oxotremorine analogs at muscarinic receptors in rat brain.

The activities of the enantiomers of BM-5 were examined to measure muscarinic cholinergic selectivity in the central nervous system. Autoradiographic studies assessed the ability of each enantiomer to inhibit the binding of [3H]-(R)-quinuclidinyl benzilate ([3H]-(R)-QNB) to muscarinic receptors in the rat brain. (+)-(R)-BM-5 inhibited [3H]-(R)-QNB binding to rat brain sections at concentrations below 1.0 microM, while 100-fold higher concentrations of (-)-(S)-BM-5 were required for comparable levels of inhibition. Analysis of the autoradiograms indicated that both stereoisomers had a similar distribution of high affinity binding sites. Each enantiomer displayed higher affinity for muscarinic receptors in the superior colliculi and lower affinity for receptors in the cerebral cortex and hippocampus. (+)-(R)-BM-5 and oxotremorine inhibited adenylyl cyclase activity in the cerebral cortex with efficacies comparable to that for acetylcholine. (+)-(R)-BM-5 was 26-fold more potent than (-)-(S)-BM-5 in inhibiting adenylyl cyclase. Oxotremorine-M and carbamylcholine stimulated phosphoinositide turnover in the cerebral cortex. Oxotremorine had lower activity and (+)-(R)-BM-5 was essentially inactive at comparable concentrations. The difference in activity of the two enantiomers indicates a remarkable stereochemical selectivity for muscarinic receptors. The stereoselectivity index is comparable for both the autoradiographic assays (48) and measures of adenylyl cyclase activity (26) in the cerebral cortex.

Okadaic acid-induced inhibition of B-50 dephosphorylation by presynaptic membrane-associated protein phosphatases.

The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.

Characterization of a glucocorticoid-sensitive hippocampal protein.

Increased synthesis of a rat hippocampal protein with an apparent molecular weight (Mr) of 35,000 Da occurs in response to elevation of serum corticosterone levels. Subcellular fractionation has localized this protein in the cytosol. Two-dimensional gel electrophoresis indicated that this protein has an isoelectric point (IEP) of 6.6. A similar protein in liver has a slightly higher Mr and an IEP of 6.8. Increased synthesis of one additional hippocampal protein with an Mr of 46,000 Da and an IEP of 6.2 and of two other liver proteins, one with an Mr of 53,000 Da and an IEP of 6.2 and the other with an Mr of 45,000 Da and a range of IEPs from 8.7 to 7.8, was also seen after injection of corticosterone into rats. One possible identity of the 35,000 Da protein is glycerol 3-phosphate dehydrogenase (GPDH), based upon the reported Mr and IEP of this enzyme. The 35,000 Da hippocampal protein co-eluted from a gel filtration column with GPDH activity. No alteration of hippocampal GPDH activity was seen in intact rats 4 or 24 h after injection of either corticosterone or the type II receptor-specific agonist RU 28362. However, daily administration of corticosterone to rats beginning 10 days after adrenalectomy returned hippocampal GPDH activity to normal values after 2-3 days. In contrast, synthesis of the 35,000 Da protein was maximally increased 4 h after a single injection of steroid and not elevated at later times.

Dephosphorylation of B-50 in synaptic plasma membranes.

Synaptic plasma membranes from rat brain cortex possess intrinsic ability to dephosphorylate the endogenous protein B-50. At low concentrations of [gamma-32P]ATP, B-50 phosphorylation in synaptic membranes is maximal at 30 seconds, followed by dephosphorylation for an additional 60 minutes. The dephosphorylation of 32P-labeled B-50 is not sensitive to the protease inhibitor leupeptin and not correlated with a loss of the B-50 content of synaptic membranes as measured with immunoblot analysis. Dephosphorylation of membrane-associated B-50 is stimulated to a small extent by Mg2+ but not by Ca2+. Heat-stable protein phosphatase inhibitors prevent dephosphorylation of 32P-labeled B-50. Dephosphorylation of B-50 in synaptic membranes is stimulated by ATP, ADP, or adenosine 5'-O-thiotriphosphate, but not by adenine, adenosine, other adenine or guanine nucleotides, nonhydrolyzable analogs of ATP or GTP, nor by adenosine 5'-O-(2-thiodiphosphate). B-50, phosphorylated by exogenous protein kinase C and purified to homogeneity, has been used as a substrate to follow the purification of B-50 phosphatase activity. B-50 phosphatase activity can be solubilized from synaptic membranes with 0.5% Triton X-100 and 75 mM KCl. Chromatography of the extract on DEAE-cellulose yields enhanced B-50 phosphatase activity.

Receptor specificity of a glucocorticoid- and stress-induced hippocampal protein.

Following treatment of rats with a subcutaneous injection of 5 mg of corticosterone, hippocampal slices in vitro show increased labeling from 35S-methionine of a protein with an apparent molecular weight (Mr) of 35,000. Increased protein labeling is seen in response to corticosterone, dexamethasone, and aldosterone, steroids that associate with glucocorticoid receptors. Little or no response occurs after administration of progesterone or estradiol. Because the injected dose of steroids is high and responses to an injection of this magnitude may be pharmacological, several experiments have been done to determine whether stimuli that increase endogenous levels of corticosterone have the same effect on labeling of the 35,000 Mr protein. One hour after various stresses (immobilization, cold, ether, and sham-injection), when plasma levels of corticosterone are elevated, labeling of the 35,000 Mr protein is increased. Injection of ACTH also stimulates the synthesis of this protein in intact animals in a manner analogous to that seen with corticosterone injections. In addition, a dose-response curve for corticosterone with adrenalectomized rats shows that synthesis of the protein is maximally increased when the injected dosage causes serum levels of corticosterone to increase to the levels seen during stress. The increase in labeling of the 35,000 Mr protein in adrenalectomized animals is only half as great as that observed in intact animals. Injections of the type II glucocorticoid (GR) receptor agonist, RU 28362, into adrenalectomized rats differentially stimulates the synthesis of the 35,000 Mr protein compared with the mineralocorticoid aldosterone, which has a higher affinity for the type I (CR) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of protein B-50 (GAP-43) from adult rat brain cortex by casein kinase II.

The phosphoprotein B-50 (GAP-43) was purified from adult rat brain cortex and phosphorylated by casein kinase II. Phosphorylation of B-50 by casein kinase II approached 1.2 mol phosphate/mol B-50. The apparent Km of casein kinase II for B-50 was 4 microM with an apparent Vmax of 13 nmol.min-1.mg-1. A tryptic phosphopeptide map on reversed phase HPLC and phosphoamino acid analysis of [32P]B-50 showed that casein kinase II phosphorylated in serine residue(s) which were located in a single tryptic peptide. Phosphorylation of B-50 by casein kinase II was inhibited more than 90% by 5 micrograms heparin/ml or 2.4 mM peptide substrate specific for casein kinase II (RRREEETEEE). The initial phosphorylation rate was increased about 2-fold by 1 mM spermine.

Characteristics of [D-Trp8]-somatostatin-sensitive B50 phosphorylation.

Inhibition of the phosphorylation of the synaptic plasma membrane (SPM) protein B50 by [D-Trp8]-somatostatin in vitro is time-dependent. Increasing the time of incubation of hippocampal synaptic plasma membranes with the peptide from 15 sec to 30 min prior to addition of 7.5 microM [gamma-32P]ATP results in a complete reduction of B50 phosphorylation. Incubation of synaptic plasma membranes for 30 min in the absence of peptide does not alter basal B50 phosphorylation. Neither ACTH nor beta-endorphin produces similar effects--inhibition of B50 phosphorylation by ACTH is maximal at 15 sec and beta-endorphin produces only a small inhibition, even after 30 min. [D-Trp8]-somatostatin is not activating a membrane-bound protease, since maximal inhibition of B50 phosphorylation by the peptide is seen in the presence of leupeptin or bacitracin. Hippocampal synaptic plasma membranes contain protein phosphatase activity. Assays of B50 phosphorylation in synaptic plasma membranes done under conditions that favor either net phosphorylation or dephosphorylation are consistent with inhibition of protein phosphatase activity by [D-Trp8]-somatostatin. This evidence suggests that [D-Trp8]-somatostatin interacts with SPM binding sites in the hippocampus, which may alter the activity of an endogenous protein phosphatase to determine the degree of B50 phosphorylation.