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The actions of somatostatin (SST) in the nervous system are mediated by specific high affinity SST receptors (SSTR1-5). However, the role of this hormone and the distribution of its receptor subtypes have not yet been defined in neural structures of the human fetus. We have analyzed four neural tissues (CNS, hypothalamus, pituitary and spinal cord) from early to midgestation for the expression of five human SSTR mRNAs, using a reverse transcription-polymerase chain reaction and Southern blot approach. These fetal neural tissues all express mRNA for multiple SSTR subtypes from as early as 16 weeks of fetal life but the developmental patterns of expression vary considerably. Transcripts for SSTR1 and SSTR2A are the most widely distributed, being expressed in all four neural tissues. SSTR2A is often the earliest transcript to be detected (7.5 weeks in CNS). SSTR3 mRNA is confined to the pituitary, hypothalamus, and spinal cord. SSTR4 is expressed in fetal brain, hypothalamus and spinal cord but not pituitary. SSTR5 mRNA is detectable in the pituitary and spinal cord by 14-16 weeks of fetal life. This mapping of SSTR mRNA expression patterns in human fetal neural tissues is an important first step toward our goal of determining the role of SST in the nervous system during early stages in human development.

OBJECTIVE To compare the expression and cellular distribution of D(2)-like dopamine receptors in the kidney of the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat. DESIGN Renal D(2)-like receptor protein expression and distribution has not been studied in the SHR. Since changes in D(2)-like receptor expression and/or distribution may contribute to the dysregulation of renal dopamine and D(1A) receptor function, we examined the expression of the three subtypes of D(2)-like receptors (D(2), D(3) and D(4)) in SHR and WKY rat kidneys. METHODS Western blot analysis and confocal immunocytochemistry with specific polyclonal antipeptide antibodies directed against the receptor subtypes, were used to assess protein expression. RESULTS There were no differences in protein expression and cellular immunolocalization of the D(2) receptor subtypes between SHR and WKY rats. Expression of the 50 kDa D(3) receptor was reduced in the cortex of the SHR; no differences in D(3) receptor levels were seen in the inner medulla of SHR and WKY rats. The D(4) receptor polypeptides were overexpressed in the cortex of SHR, while in the inner medulla no difference in expression of the D(4) receptor proteins was observed between SHR and WKY rats. Immunocytochemistry also showed increased immunostaining of D(4) receptors in tubular structures in the cortex, but diminished staining in the SHR inner medulla. CONCLUSION The observed differences in expression and distribution of D(3) and D(4) dopamine receptors between cortex and inner medulla of the kidneys of SHR and WKY rats may contribute to the aberrant state of dopaminergic-mediated natriuresis in SHR.

Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.

Pulsatile growth hormone (GH) secretion differs between males and females and regulates the sex-specific expression of cytochrome P450s in liver. Sex steroids influence the secretory dynamics of GH, but the neuroendocrine mechanisms have not been conclusively established. Because periventricular hypothalamic somatostatin (SST) expression is greater in males than in females, we generated knockout (Smst-/-) mice to investigate whether SST peptides are necessary for sexually differentiated GH secretion and action. Despite marked increases in nadir and median plasma GH levels in both sexes of Smst-/- compared with Smst+/+ mice, the mutant mice had growth curves identical to their sibling controls and retained a normal sexual dimorphism in weight and length. In contrast, the liver of male Smst-/- mice was feminized, resulting in an identical profile of GH-regulated hepatic mRNAs between male and female mutants. Male Smst-/- mice show higher expression of two SST receptors in the hypothalamus and pituitary than do females. These data indicate that SST is required to masculinize the ultradian GH rhythm by suppressing interpulse GH levels. In the absence of SST, male and female mice exhibit similarly altered plasma GH profiles that eliminate sexually dimorphic liver function but do not affect dimorphic growth. Fil: Low, Malcolm J.. Oregon Health And Science University; Estados Unidos Fil: Otero Corchon, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Oregon Health And Science University; Estados Unidos Fil: Parlow, Albert. F.. University of California at Los Angeles; Estados Unidos Fil: Ramirez, Jose L.. McGill University; Canadá Fil: Kumar, Ujenda. McGill University; Canadá Fil: Patel, Yogesh C.. McGill University; Canadá Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

BACKGROUND We have analysed the distribution of the five somatostatin receptors (sst1–5) by immunohistochemistry in a large retrospective series of 51 medullary carcinoma of the thyroid (MCT) specimens and correlated the pattern of sst expression with expression of somatostatin (SRIF) peptide, tumour pathology and clinical outcome. MEASUREMENTS Immunohistochemistry was performed with rabbit polyclonal antipeptide antibodies directed against the extracellular domains or cytoplasmic tail of human (h) sst1–5. SRIF immunoreactivity was investigated in parallel paraffin sections. RESULTS Eighty-five percent of the tumours were positive for one or more sst, localized to both tumour cells as well as surrounding peritumoural structures, especially blood vessels. Forty-nine percent of the tumours were positive for sst1, 43% for sst2, 47% for sst3, 4% for sst4, and 57% for sst5. Fifty-one percent of tumours expressed one or two sst subtypes; 33% were positive for three or more sst isoforms. All five sst receptors were detected in only two cases. Tumours expressing octreotide sensitive subtypes (sst2,3,5) accounted for 75% of the series. 50% of the tumours co-expressed SRIF suggesting tumour cell regulation by endogenous SRIF via paracrine/autocrine circuits. There was no correlation between sst1–5 expression and age, sex, tumour size or stage, histological type or clinical outcome. Simultaneous analysis of primary tumour and lymph node metastases revealed a similar pattern of sst immunoreactivity indicating that sst expression is not modified in the course of disease progression. CONCLUSIONS With the exception of sst4, medullary carcinoma of the thyroid display a rich but heterogeneous expression of sst subtypes. Immunohistochemical typing of sst receptor expression using specific antireceptor antibodies represents an ideal approach for characterizing sst subtype expression in medullary carcinoma of the thyroid for optimizing receptor targeted diagnosis and therapy with somatostatin analogs.

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.

Niemann–Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1 , a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino–cupric–silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2- to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent NH 4 Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.

We have developed a panel of rabbit polyclonal antipeptide antibodies against the five human somatostatin receptor subtypes (hSSTR1-5) and used them to analyze the pattern of expression of hSSTR1-5 in normal human islet cells by quantitative double-label confocal fluorescence immunocytochemistry. All five hSSTR subtypes were variably expressed in islets. The number of SSTR immunopositive cells showed a rank order of SSTR1 > SSTR5 > SSTR2 > SSTR3 > SSTR4. SSTR1 was strongly colocalized with insulin in all beta-cells. SSTR5 was also an abundant isotype, being colocalized in 87% of beta-cells. SSTR2 was found in 46% of beta-cells, whereas SSTR3 and SSTR4 were relatively poorly expressed. SSTR2 was strongly colocalized with glucagon in 89% of alpha-cells, whereas SSTR5 and SSTR1 colocalized with glucagon in 35 and 26% of alpha-cells, respectively. SSTR3 was detected in occasional alpha-cells, and SSTR4 was absent. SSTR5 was preferentially expressed in 75% of SST-positive cells and was the principal delta-cell SSTR subtype, whereas SSTR1-3 were colocalized in only a few delta-cells, and SSTR4 was absent. These studies reveal predominant expression of SSTR1, SSTR2, and SSTR5 in human islets. Beta-cells, alpha-cells, and delta-cells each express multiple SSTR isoforms, beta-cells being rich in SSTR1 and SSTR5, alpha-cells in SSTR2, and delta-cells in SSTR5. Although there is no absolute specificity of any SSTR for an islet cell type, SSTR1 is beta-cell selective, and SSTR2 is alpha-cell selective. SSTR5 is well expressed in beta-cells and delta-cells and moderately well expressed in alpha-cells, and thereby it lacks the islet cell selectivity displayed by SSTR1 and SSTR2. Subtype-selective SSTR expression in islet cells could be the basis for preferential insulin suppression by SSTR1-specific ligands and of glucagon inhibition by SSTR2-selective compounds.

We have investigated the role of the cytoplasmic tail (C-tail) of the human somatostatin receptor type 5 (hSSTR5) in regulating receptor coupling to adenylyl cyclase (AC) and in mediating agonist-dependent desensitization and internalization responses. Mutant receptors with progressive C-tail truncation (Delta347, Delta338, Delta328, Delta318), Cys320 --> Ala substitution (to block palmitoylation), or Tyr304 --> Ala substitution of a putative NPXXY internalization motif were stably expressed in Chinese hamster ovary K1 cells. Except for the Tyr304 --> Ala mutant, which showed no binding, all other mutant receptors exhibited binding characteristics (Kd and Bmax) and G protein coupling comparable with wild type (wt) hSSTR5. The C-tail truncation mutants displayed progressive reduction in coupling to AC, with the Delta318 mutant showing complete loss of effector coupling. Agonist pretreatment of wt hSSTR5 led to uncoupling of AC inhibition, whereas the desensitization response of the C-tail deletion mutants was variably impaired. Compared with internalization (66% at 60 min) of wt hSSTR5, truncation of the C-tail to 318, 328, and 338 residues reduced receptor internalization to 46, 46, and 23%, respectively, whereas truncation to 347 residues slightly improved internalization (72%). Mutation of Cys320 --> Ala induced a reduction in AC coupling, desensitization, and internalization. These studies show that the C-tail of hSSTR5 serves a multifunctional role in mediating effector coupling, desensitization, and internalization. Whereas coupling to AC is dependent on the length of the C-tail, desensitization and internalization require specific structural domains. Furthermore, internalization is regulated through both positive and negative molecular signals in the C-tail and can be dissociated from the signaling and acute desensitization responses of the receptor.

The human somatostatin receptor subtype 5 (hSSTR5) gene has previously been cloned and localized to chromosome 16 p13.3. This region is evolutionarily conserved in all vertebrate genomes from the puffer fish (Fugu rubripes) to human, and also contains loci for genes associated with two common multisystemic disorders, adult polycystic kidney disease (PKD1) and tuberous sclerosis (TSC2). Analysis of the 5′ flanking region of the hSSTR5 gene has revealed consensus sequences for a number of transcription factors as well as Alu-like repeat elements. In the present study, genomic DNA from 53 unrelated individuals was analysed by PCR and Southern blots probed with radiolabeled fragments generated from different segments of the hSSTR5 gene. We have identified two restriction fragment length polymorphisms (RFLP) with high heterozygosity values at the 5′ flanking region of the hSSTR5 gene. These RFLP markers will be useful for determining the allelic loss of genetic material from this region. The observed polymorphism in the promoter region may affect the function of the hSSTR5 gene.

The ligand binding domain of G protein-coupled receptors for peptide ligands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, previous studies have suggested that the binding cavity of the octapeptide analog SMS201-995 (SMS) is lined by residues in TMs III-VII. The additional involvement of the extracellular domains for binding SMS or the natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Using a cassette construct cDNA for the human somatostatin 5 receptor (sst5R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellular portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for the NH2-terminal segment) or truncation analysis (ECL3). CHO-K1 cells were stably transfected with wild type or mutant human sst5R constructs, and agonist binding was assessed using membrane binding assays with 125I-LTT SRIF-28 ligand. Deletion of the NH2 terminus or CSE mutagenesis of ECL1 and ECL3 produced minor 2-8-fold decreases in affinity for SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the size of this loop in sst1R and sst4R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF-28. In contrast, both ECL2 mutants failed to bind 125I-LTT SRIF-28. Immunocytochemical analysis of nonpermeabilized cells with a human sst5R antibody revealed that the mutant receptors were targeted to the plasma membrane. Labeled SMS (125I-Tyr3 SMS) also failed to bind to the mutant ECL2 receptors. These results suggest a potential contribution of ECL2 (in addition to the previously identified residues in TMs III-VII) to the SRIF ligand binding pocket.

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

We have characterized the biosynthetic origin of somatostatin-14 (SS-14), SS-28, and pro-SS [1-10] from pro-SS (PSS) in 1027B 2 rat islet tumor cells. Because these cells lack regulated secretion and show unresponsiveness of the SS gene to cAMP, we have additionally carried out morphological and functional studies to elucidate the molecular defect in cAMP signalling and to localize the sites of PSS maturation along the secretory pathway. Cell extracts and secretion media were analysed by high performance liquid chromatography and specific C- and N-terminal radioimmunoassays. Electron microscopic sampling of 1027B 2 cell cultures showed that most cells had very few dense core secretory granules for heterogeneous sizes. The cells expressed the endoproteases furin, PC1, and PC2 and contained large quantities of fully processed SS-14 and SS-28 with very little unprocessed PSS (ratio SS-14:SS-28:PSS=39:51:10%). They secreted high concentrations of SS-14, SS-28, and PSS [1-10] constitutively along with PC1 and PC2. Pulse-chase studies demonstrated that PSS is rapidly (within 15 min), and efficiently processed to SS-14, SS-28, and PSS [1-10] via separate biosynthetic pathways: PSS→SS-14+8 kDa; PSS→SS-28+7 kDa; PSS→PSS [1-10] . Monensin reduced intracellular SS-like immunoreactivity without altering processing efficiency. Transfection with the catalytic subunit of protein kinase A (PKA-C) activated SS promoter-CAT activiting indicating that the defect in cAMP-dependent signaling in 1027B 2 cells lies at the level of PKA-C. PKA-C overexpression failed to alter the ratio of processed SS-14 and SS-28. These results demonstrate that SS-14, SS-28, and PSS [1-10] are independently synthesized from PSS and that efficient precursor processing can occur within the constitutive secretory pathway in the relative absence of dense core secretory vesicles.

Somatostatin (SST) exerts direct antiproliferative effects in tumor cells, triggering either growth arrest or apoptosis. The cellular actions of SST are transduced through a family of five distinct somatostatin receptor subtypes (SSTR1-5). Whereas growth inhibition has been reported to follow stimulation of protein tyrosine phosphatase via SSTR2 or inhibition of Ca2+ channels via SSTR5 in heterologous expression systems, the subtype selectivity for signaling apoptosis has not been investigated. The tumor suppressor protein p53 and the protooncogene product c-Myc regulate cell cycle progression (growth factors present) or apoptosis (growth factors absent). The p53-induced G1 arrest requires induction of p21, an inhibitor of cyclin-dependent kinases, whereas apoptosis requires induction of Bax. c-Myc is capable of abrogating p53-induced G1 arrest by interfering with the inhibitory action of p21 on cyclin-dependent kinases. We have, therefore, investigated the regulation of p53, p21, c-Myc, and Bax and cellular apoptosis in relation to cell cycle progression in CHO-K1 cells stably expressing individual human SSTR1-5. We demonstrate that apoptosis is signaled uniquely through human SSTR3 and is associated with dephosphorylation-dependent conformational change in wild-type (wt) p53 as well as induction of Bax. The induction of wt p53 occurs rapidly and precedes the onset of apoptosis. We show that the increase in wt p53 is not associated with the induction of p21 or c-Myc when octreotide-induced apoptosis becomes evident, suggesting that such apoptosis does not require G1 arrest and is not c-Myc dependent. These findings provide the first evidence for hormonal induction of wt p53-associated apoptosis via G protein-coupled receptor in a subtype-selective manner.

Somatostatin (SRIF) receptors (ssts) comprise a family of heptahelical membrane proteins encoded by five related genes that map to separate chromosomes and which, with the exception of sst 1 , are intronless. The ssts 1–4 display weak selectivity for SRIF-14 binding, whereas sst 5 is SRIF-28—selective. Based on structural similarity and reactivity for octapeptide and hexapeptide sst analogs, ssts 2,3 and sst 5 belong to a similar sst subclass; ssts 1–4 react poorly with these analogs and belong to a separate subclass. All five ssts are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin-sensitive guanosine triphosphate (GTP)-binding proteins. mRNA for ssts 1–5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective and tissue- and species-specific. All pituitary cell subsets express sst 2 and sst 5 , with sst 5 being more abundant. Individual pituitary cells coexpress multiple sst subtypes. The binding pocket for SRIF-14 ligand lies deep within the membrane in transmembrane domains (TMDs) 3 to 7. Except for extracellular loop 2, it does not involve the other exofacial structures. Human (h)sst 2 A and hsst 5 undergo agonist-mediated desensitization, associated with receptor internalization. The C-tail segment of hsst 5 displays positive molecular internalization signals. The ssts inhibit the growth of tumor cells directly, through blockade of mitogenic signaling leading to growth arrest and through induction of apoptosis. This process is associated with translocation of phosphotyrosine phosphatase (PTP) 1C from the cytosol to the membrane.

The role of PC2 in prosomatostatin (PSS) processing was investigated in GH3/GH4C1 pituitary cells. These cells are sparsely granulated, express different amounts of PC2 and no PC1. We described heterologous processing of rat PSS (rPSS) co-expressed with PC2 in stably transfected cells, correlate PC2 protein levels under different conditions of transfection with efficiency of PSS processing to somatostatin-14 (SS-14), determine the effect of modulating cell granularity on enzyme expression and PSS processing, and compare the relative potency of PC2 with that of PC1, PSS and cleavage products were monitored by HPLC and radioimmunoassay of SS-like immunoreactivity (SSLI). Radioimmunoassay analysis of N-terminal PC2-like immunoreactivity (PC2 LI) in GH4C1:rPSS, GH4C1:rPSS + PC2 and GH3:rPSS transfectants showed a gradient of PC2 protein of 1:2.6:3.4 in cell extracts and 1:4.7:9 in secretion media from these cells respectively. The concentration of PC2 protein correlated with SS-14 conversion efficiency was 36 +/- 3% in GH4C1:rPSS cells, 56 +/- 7% in GH4C1:rPSS-PC2 cells and 100% in GH3:rPSS cells. Treatment of GH4C1:rPSS + PC2 cells with epidermal growth factor, insulin, and beta-estradiol to induce granules, significantly increased basal and forskolin-stimulated co-release of SS LI and PC2 LI, but had no influence on SS-14 processing efficiency. Hormone treatment led to a small increase in the ratio of mature PC2 (68 kDa) to proPC2 (75 kDa) forms. PC1 stably transfected in GH4C1 cells produced significantly greater SS-14 conversion (62% in cells, 66% in media) compared with PC2 transfectants (53% in cells, 47% in media) These results provide the first proof that PC2 can effect dibasic processing of mammalian PSS, and, along with PC1, qualifies as an authentic SS-14 convertase. The activity of PC2 requires the milieu of the secretory cell but not the secretory granule.

We have previously reported that rat prosomatostatin (rPSS) undergoes conversion at Arg decreases and Lys decreases monobasic sites to SS-28 and PSS-(1-10) respectively in COS-7 cells, and have proposed furin or a related enzyme of the constitutive secretory pathway as the endoproteinase responsible. Here we have tested directly the ability of furin to cleave rPSS at the two monobasic sites as well as at the RXRK dibasic site of SS-14 conversion (a furin motif, except for Lys substituting for Arg at P1). Recombinant vaccinia virus (VV) vectors were used to co-express rPSS with graded doses of furin in COS-7 cells and LoVo colon carcinoma cells deficient in furin. PSS and cleavage products in cell extracts and media were characterized by HPLC analysis and C-terminal [SS-14-like immunoreactivity (SS-14 LI)] and N-terminal [PSS-(1-10) LI] directed radioimmunoassays. There was a dose-dependent increase in SS-28 production from rPSS by furin in COS-7 cells from 29% (control) to 58% (high-dose furin) associated with a progressive decrease in unprocessed PSS from > 60% to approximately 20% of total SS-14 LI. Significant SS-14 production occurred only at high levels of furin infection. Control LoVo cells infected with VV:rPSS exhibited production of approximately 21% SS-28, approximately 15% PSS-(1-10) and 3.5% SS-14. Infection of LoVo cells with VV:hfurin (hfurin = human furin) enhanced SS-28 production to 30-34%. SS-14 synthesis also increased to 25-40%, probably by conversion from SS-28. Overexpression of furin in COS-7 or LoVo cells failed to increase PSS-(1-10) production. These results show that furin is a candidate SS-28 convertase. Arginine is the preferred residue at the P1 site of furin cleavage. Furin does not process rPSS to PSS-(1-10), suggesting the existence of another monobasic convertase with a preference for Lys rather than Arg at P1. Such an enzyme could also explain the presence of endogenous SS-28-, PSS-(1-10)- and SS-14-producing activities in LoVo cells.

Expression of the lipophilic ligand transporter, apolipoprotein D (apoD) by primary astrocyte cultures derived from neonatal mouse brain was investigated. Western blot analysis of cell lysates and media showed that apoD is constitutively secreted by astrocytes with little intracellular storage. The secreted apoD floated primarily at density 1.063-1.21 g ml -1 upon sequential ultracentrifugation indicating its association with lipids. Treatment of astrocytes with the carboxylic ionophore, monensin, resulted in intracellular retention and decreased secretion of apoD that was of slightly reduced M r . Progesterone, a steroid hormone that binds to apoD with high affinity (10 -6 mol l -1 ) and the oxysterol, 25-hydroxycholesterol which is a potent regulator of cellular cholesterol homeostasis in mammalian cells, differentially stimulated apoD, but not apoE secretion. These results show that astrocytes synthesize and constitutively secrete apoD and suggest a physiologic role for this lipocalin in cholesterol metabolism in the nervous system

Expression of mRNA for hSSTR1-5 was determined in secretory (GH, PRL, TSH, ACTH) and nonsecretory pituitary tumors, as well as normal human fetal and adult pituitary by reverse transcriptase (RT) PCR followed by Southern blots. All 5 hSSTR subtype mRNAs were expressed in fetal pituitary, while adult pituitary was positive for 4 subtypes, lacking hSSTR4 mRNA. All 15 tumors analyzed were positive for SSTR mRNA, 14 expressing more than one subtype. SSTR2 mRNA in all tissues was expressed as the 2A variant, there being no detectable transcript for SSTR2B. Amongst the 5 SSTRs, mRNA for SSTR2A was the most frequently expressed (87% of tumors) followed by SSTR1 (73%), SSTR3 (53%), SSTR5 (47%), and SSTR4 (40%). The frequency and pattern of expression of the SSTR mRNAs was virtually identical in the different tumor subclasses and did not correlate with tumor size. Since pituitary tumors are monoclonal in origin, multiple SSTR genes are expressed in individual cells. Most tumors are rich in SSTR1 and SSTR2A mRNA compared to the other subtypes. This implies that SST analogs like SMS 201-995, known to interact with SSTR2A, but not with SSTR1, act on pituitary tumors mainly via the SSTR2 subtype.

Recent reports have suggested that only some of the cloned somatostatin receptors (SSTRs) are coupled to adenylyl cyclase. These studies have used both stable and transiently transfected cells or cells lacking appropriate Gi alpha and are controversial. To investigate SSTR signalling mechanisms, we have established stably transfected CHO-K1 cells expressing human genes for SSTR1-5. The effect of 0.1-100 nM SST-14 and SST-28 on forskolin (1 microM) stimulated cAMP accumulation was determined and compared to their receptor binding affinities. The 5 expressed hSSTRs bound SST-14 and SST-28 with high affinity (IC50 1.1-2.1 nM for SST-14; IC50 0.25-5.4 nM for SST-28). hSSTR1-4 bound SST-14 > SST-28 whereas hSSTR5 bound SST-28 > SST-14. Radioligand binding to hSSTR1-5 was significantly inhibited by GTP, GTP gamma S and pertussis toxin. Both SST-14 and SST-28 inhibited forskolin-induced cAMP stimulation with ED50 values which paralleled their binding affinities for the individual hSSTR subtypes. These results demonstrate that all 5 human SSTRs are functionally coupled to inhibition of adenylyl cyclase in CHO-K1 cells via pertussis toxin sensitive G proteins.

The drug cysteamine (CHS) induces a profound loss of somatostatin-14 (SS-14) biological and immunological (SS-14 LI) activity from somatostatin cells in vivo and in vitro. The present study was designed to determine (i) whether CHS induced loss of somatostatin is accompanied by secondary increases in SS-mRNA perhaps through loss of autoinhibition of somatostatin cells; (ii) whether CHS exerts additional direct effects on SS gene regulation. CHS was administered to rats in vivo or applied in vitro to primary cultures of rat islet cells, rat islet somatostatin-producing tumor cells (1027 B 2 ), and endogenous or in vitro synthesized SS-mRNA. In vivo administration of CHS led to 80% reduction in tissue SSLI by 4 h. These changes were accompanied by significant alterations in SS-mRNA that were both tissue-specific and time-dependent. The pattern in brain and intestine was typified by a significant 60% increase in SS-mRNA at 2 h followed by a gradual reduction to ∼55% of control at 8 h. Stomach showed a significant 95% increase in SS-mRNA at 4 h followed by a 37% decrease by 8 h. Pancreatic SS-mRNA displayed a sustained 25–65% reduction for 8 h. Pretreatment of islet cell cultures with CHS reproduced the in vivo findings with pancreas viz. decreased SSLI (80–90% of control) accompanied by a parallel reduction in SS-mRNA (40–50% of control) sustained from 2–72 h. CHS also induced a reduction in immunoreactive insulin and insulin mRNA in cultured islet cells. As with normal islet cells, CHS treatment of 1027 B 2 islet tumor cells led to a profound and sustained decrease in SSLI and SS-mRNA. These changes occurred in the absence of any alteration in intracellular cAMP levels. CHS was without effect when incubated directly with SS-mRNA isolated from 1027 B 2 cells or with in vitro synthesized SS-mRNA. We conclude that in addition to its effect on SSLI, CHS also induces time- and tissue-dependent alterations in SS-mRNA. The mechanism of CHS action on SS-mRNA is complex and may involve both an indirect effect secondary to loss of somatostatin autoinhibition (to account for SS-mRNA increases) and/or a direct inhibition of SS gene expression (to explain SS-mRNA reduction). The precise site of direct CHS action on SS gene regulation remains to be defined. Since CHS inactivates cellular somatostatin and produces longterm reductions in SS-mRNA in all tissues, it represents a useful tool for inducing functional lesions of somatostatin cells at both gene and peptide levels.

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and exhibits SS-14-and SS-28-selective subtypes. Whether such subtypes arise from molecular heterogeneity of the receptor protein has not been definitively established. Previous reports characterizing the molecular properties of the SS receptor by the cross-linking approach have yielded divergent size estimates ranging from 27 kDa to 200 kDa. In order to resolve this discrepancy, as well as to determine whether SS-14 and SS-28 interact with specific receptor proteins, we have cross-linked radioiodinated derivatives of [125I-Tyr11]SS-14 (T*-SS-14) and [Leu8,D-Trp22,125I-Tyr25]SS-28 (LTT*-SS-28) to membrane SS receptors in rat brain, pituitary, exocrine pancreas and adrenal cortex using a number of chemical and photoaffinity cross-linking agents. The labelled cross-linked receptor proteins were analysed by SDS/PAGE under reducing conditions followed by autoradiography. Our findings indicate that the pattern of specifically labelled cross-linked SS receptor proteins is sensitive to the concentration of chemical cross-linking agents such as disuccinimidyl suberate and dithiobis-(succinimidyl propionate). Labelled high-molecular-mass complexes of cross-linked receptor-ligand proteins were observed only when high concentrations of these cross-linkers were employed. Using optimized low concentrations of cross-linkers, however, two major labelled bands of 58 +/- 3 kDa and 27 +/- 2 kDa were detected. These two bands were identified as specifically labelled SS receptor proteins subsequent to cross-linking with a number of photoaffinity cross-linking agents as well. We demonstrate here that the 58 kDa protein is the major SS receptor protein in the rat pituitary, adrenal and exocrine pancreas, whereas the 27 kDa moiety represents the principal form in the brain. Additionally, the presence of a minor specifically labelled band of 32 kDa was detected uniquely in the brain, and a minor labelled protein of 42 kDa was observed in the pancreas. The labelling pattern obtained with LTT*-SS-28 was identical to that observed with T*-SS-14. Labelling of the 27 kDa band by either ligand was inhibited by SS-14 and SS-28 in a dose-dependent manner. Densitometric quantification showed that SS-14 exhibited greater than 2-fold greater potency than SS-28 for inhibiting the labelling of the 27 kDa species. These findings emphasize the need for careful interpretation of cross-linking data obtained for SS receptors, and provide evidence for molecular heterogeneity and for a tissue-specific distribution of the two principal SS receptor proteins.

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphon-ovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p < 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA. These increases were selective for SS mRNA and were not accompanied by any change in β-actin mRNA. By contrast, glutamate at the single dose tested was without effect on both SSLI and SS mRNA. These results demonstrate that Quin induces a selective NMDA receptor-mediated stimulation of SS gene expression and SS biosynthesis in vitro and suggest a similar mechanism for the augmented striatal SSLI in HD.

Mammalian prosomatostatin (pro-S) undergoes extensive processing at the C-terminal segment where the somatostatin-like biological activities (S-14 and S-28) reside. The recent discovery of pro-S-(1-10) (antrin) as a prominent mature product in the stomach suggests that pro-S may also be processed at the N-terminus. In the present study we have developed an antibody directed against the N-terminal segment of pro-S-(1-10) capable of detecting peptides extended at the C-terminus of pro-S-(1-10) to characterize N-terminal processing of rat pro-S. Specifically, we have 1) examined the relative abundance of pro-S-(1-10)-like immunoactivity [pro-S-(1-10)] in different somatostatin tissues as an index of tissue-specific N-terminal processing, 2) compared the concentrations of pro-S N- and C-terminal immunoreactive peptides, 3) used HPLC and region-specific RIAs directed against both the N- and C-terminal segments of pro-S to identify and characterize novel N-terminal peptides, 4) studied the tissue distribution and release of the N-terminal peptides; and 5) characterized and quantified a 7-kDa molecule equivalent to pro-S without the C-terminal S-28 sequence. Acetic acid (1 M)-pepstatin extracts of hypothalamus, cerebral cortex, antrum, jejunal mucosa, and pancreas were fractionated by reverse phase and gel permeation HPLCs. Whole tissue extracts as well as the column effluent were monitored by region-specific RIAs using antibodies against pro-S-(1-10), S-28-(1-12), and S-14. Other than the pancreas, all S-producing tissues were rich in pro-S-(1-10) LI. Its concentration was 1- to 4-fold lower than those of S-14 LI and S-28-(1-12) LI. Tissue pro-S-(1-10) LI was heterogeneous, consisting of at least eight molecular forms with respective mol wt of 1,000 (1 kDa), 1,500 (1.5 kDa), 2,500 (2.5 kDa), 3,500 (3.5 kDa), 4,500 (4.5 kDa), 7,000 (7 kDa), 8,000 (8 kDa), and 10,000 (10 kDa). Based on the simultaneous presence or absence of C-terminal immunoreactivity, the 10-kDa form corresponded to pro-S, 8 kDa to pro-S-(1-76), and 7 kDa to pro-S without the S-28 sequence. The predominant N-terminal forms corresponded to 1 kDa [pro-S-(1-10)] and 7 kDa. The 1-, 1.5-, 2.5-, and 7-kDa forms were identified as secretion products in portal blood or in medium from cultured 1027 B2 islet somatostatin cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Prosomatostatin is cleaved at dibasic and monobasic sites to produce somatostatin-14 and somatostatin-28 respectively. The mammalian pro-protein convertases comprising furin, PACE4 and PC1-6 have recently been identified and are believed to mediate endoproteolysis of prohormone precursors such as prosomatostatin. Furin is membrane bound, localized to the Golgi and mediates constitutive processing. PC1 and PC2 are soluble and are expressed solely in endocrine and neuroendocrine tissues suggesting a key role in prohormone processing. We have investigated the endogenous and heterologous synthesis and processing of rat prosomatostatin in 1027B2 rat islet somatostatinoma cells and in constitutive (COS-7, PC-12) and regulated (AtT-20, GH3/GH4C1) secretory cells. We have correlated processing efficiency with: secretion through the constitutive or regulated pathways; endogenous expression of furin, PC1 and PC2; and expression or overexpression of furin, PC1 and PC2. Pulse-chase studies showed that prosomatostatin is rapidly and independently processed to somatostatin-14 and somatostatin-28. Furin is capable of monobasic processing of prosomatostatin and is a candidate somatostatin-28 convertase. PC1 and PC2 both effect dibasic processing of prosomatostatin and qualify as putative somatostatin-14 convertases. PC1 is active in constitutive and regulated secretory cells, has a broader specificity and is overall more potent than PC2. Efficient processing of prosomatostatin begins in a Golgi or pre Golgi compartment. It requires the milieu of the secretory cell but not the secretory granule.

The intense interest in SST as a model to study neuropeptide synthesis has provided a framework for understanding the biosynthetic pathways, the putative role of PCs, and the secretory pathways required for PSST maturation to its active products. Kinetic studies have demonstrated that PSST is rapidly and independently processed to SST-14, SST-28, and PSST[1-10]. Both PC1 and PC2 are capable of processing PSST to SST-14. Furin and PACE 4 effect monobasic cleavage and are candidate SST-28 convertases. Contrary to previous belief, cleavage at the NH2-terminus has been shown not require the monobasic Lys13 residue with SKI-1 acting as the most likely convertase. Secretory granules are not a requirement for PSST maturation. Additionally, the conserved amino terminal segment of PSST serves as a sorting for the regulated secretory pathway. Beyond the pure biochemical and basic research interest in defining the specific intracellular events implicated in processing and sorting of PSST, combining the knowledge gained from the processing and targeting aspects to treatments of various diseases would be the ultimate satisfaction of any scientist. Such an approach was elegantly attempted by Rivera and colleagues who used the ER as the storage compartment for genetically engineered secretory proteins such as insulin and growth hormone (53).

In kidney, nitric oxide (NO) synthesized by nitric oxide synthase (NOS) regulates sodium and water excretion, and renal medullary blood flow. The expression of constitutive NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS), were assessed in kidney of the spontaneously hypertensive rat (SHR) and the normotensive Wistar Kyoto (WKY) rat by Western blot analysis and immunocytochemistry. Neuronal NOS expression was observed in the cortex and eNOS was detected only in theinner medulla of both WKY and SHR. In SHR, expression of eNOS was attenuated to 35.1 +/- 10.8%, while expression of nNOS was only 57.5 +/- 5.7% of the levels seen in WKY rat. Immunocytochemical studies revealed decreased staining of nNOS in the macula densa, collecting ducts and in the glomerulus of SHR compared to WKY rat. Endothelial NOS immunoreactivity was restricted to vascular structures of the inner intima cells and smooth muscle cells, and was markedly reduced in the vasculature of SHR. The decreased renal blood flow observed in SHR may be linked to a diminished expression of eNOS and nNOS, underscoring the importance of these enzymes in the pathophysiology and maintenance of genetic hypertension.

The peptide somatostatin (SST) is widely synthesized in the brain and periphery and acts through a family of five receptors (SSTR1-5) to exert numerous effects. A gene product related to SST, cortistatin (CST), also interacts with SSTR1-5. Here we have investigated the regulation of SSTR1-5 and of CST in SST knockout (SSTKO) mice. The five SSTRs were quantitated individually by subtype-selective binding analysis, by immunocytochemistry, and by mRNA measurement and showed, in the brain of SSTKO mice, up-regulation of subtypes 1, 2, 4, and 5, and down-regulation of SSTR3. Peripheral tissues displayed both subtype- and tissue-specific changes in SSTR1-5 mRNA levels of expression. Lack of SST did not up-regulate normal CST expression in brain nor did it induce its expression in the periphery. SST-like immunoreactivity, however, was induced in the proximal midgut in SSTKO animals, suggesting intestinal expression of a novel SST-like gene. Fil: Ramírez, José L.. McGill University; Canadá Fil: Mouchantaf, Rania. McGill University; Canadá Fil: Kumar, Ujendra. McGill University; Canadá Fil: Otero Corchon, Veronica. Oregon Health and Science University; Estados Unidos Fil: Rubinstein, Marcelo. Oregon Health and Science University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Low, Malcolm J.. Oregon Health and Science University; Estados Unidos Fil: Patel, Yogesh C.. McGill University; Canadá

Hepatic stellate cells (HSCs) are considered therapeutic targets to decrease portal hypertension. To elucidate some of the hemodynamic effects of somatostatin (SST) on portal pressure, the presence and function of SST receptors (SSTRs) on HSCs were investigated.SSTR messenger RNA expression, and SSTR presence was investigated using reverse-transcription polymerase chain reaction, real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The function of SSTRs was studied by examining the effects of SST and specific SSTR agonists on endothelin-1(ET-1)-induced HSC contraction.Specific amplicons for SSTR subtypes 1, 2, and 3 were demonstrated in rat liver and in activated HSCs. The presence of SSTR subtypes 1, 2, and 3 was confirmed by Western blotting. With immunohistochemistry, a strong staining of HSCs was obtained for SSTR subtypes 1, 2, and 3 in CCl4-treated rats, but not in normal rat liver. Incubation of HSCs on collagen gels with buffer, 10(-8) mol/L SST, and 2 x 10(-8) mol/L ET-1 resulted in collagen surface area decreases of 5.5% +/- 3.3%, 6.8% +/- 4.4%, and 49.8% +/- 8.3%, respectively. Relative contraction of gels preincubated with 10(-8) mol/L SST followed by 2 x 10(-8) mol/L ET-1 or vice versa as compared with maximal contraction (100%) with 2 x 10(-8) mol/L ET-1 were 72.6% +/- 17.9% and 76.2% +/- 12.6%, respectively (P0.05). SSTR agonist 1, but not SSTR agonist 2 or 3, was able to counteract the contractile effect of ET-1. CONCLUSIONA: Activated rat HSCs bear SSTR subtypes 1, 2, and 3. SST causes significant partial inhibition of ET-1-induced contraction of activated HSCs, mainly by stimulation of SSTR subtype 1.

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH(2)-terminal segment of PSST consists of an amphipathic alpha-helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3--15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3--15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro(5)--Gln(12) as being important in precursor targeting, with Leu(7) and Leu(11) being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha-helix. Disruption of the alpha-helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.

The twelve tasks comprising this four year project were completed and a' full report submitted in September 2000. However, in the course of these studies three new leads were pursued as three separate tasks (Tasks 13, 14, 15) which were not part of the original contract but because of their importance were nonetheless developed substantially and also reported upon in September 2000. We then requested and received approval for a no-cost extension to allow us to round off these three new tasks up to the period ending September 2001. We made reasonable progress in each of these three areas as outlined in this report despite the time and budgetary constraints and personnel departures. As emphasized in our Final Report, however, each of these three new tasks represented substantial new projects which we intend to pursue fully with renewed new longterm funding.

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.

Preprocortistatin (PPCST) has been recently identified as a novel somatostatin (SST)-related gene expressed only in brain. PPCST shares 11 of 14 residues with SST-14 at its C-terminal segment, where it features Lys-Lys and Lys-Arg basic sites for cleavage to putative cortistatin (CST)-14 and CST-29 peptides, respectively. Although synthetic replicates of the two putative CST peptides interact with SST receptors, they also display novel effects suggesting independent biological functions. Nothing is currently known about the naturally occurring mature cleavage products of PPCST posttranslational processing. Here we have cloned rat PPCST cDNA, stably expressed it in AtT-20 pituitary cells, and characterized the cellular and releasable products of PPCST processing by HPLC and radioimmunoassay using a SST-14 antibody that recognizes synthetic CST-14 and CST-29. Transfected cells released 120 +/- 21 pg of total CST-LI per plate basally, with an increase to 204 +/- 33 pg per plate with forskolin stimulation (p < 0.05). HPLC chromatograms of cell extracts revealed three peaks corresponding to CST-14, CST-29, and unprocessed PPCST (ratio, 41:55:4.5). CST was released preferentially as CST-14 (63-70%) compared with CST-29 (30-37%) under basal and forskolin-stimulated conditions. These studies demonstrate efficient processing of PPCST to both CST-14 and CST-29 through putative cleavage at both C-terminal dibasic sites of PPCST. Although the two peptides are synthesized approximately equally, CST-14 is released preferentially via the regulated secretory pathway.

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.

Somatostatin (SST), a regulatory peptide, is produced by neuroendocrine, inflammatory, and immune cells in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide is released in large amounts from storage pools of secretory cells, or in small amounts from activated immune and inflammatory cells, and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors that comprise five distinct subtypes (termed SSTR1-5) that are endoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. Short synthetic octapeptide and hexapeptide analogs bind well to only three of the subtypes, 2, 3, and 5. Selective nonpeptide agonists with nanomolar affinity have been developed for four of the subtypes (SSTR1, 2, 3, and 4) and putative peptide antagonists for SSTR2 and SSTR5 have been identified. The ligand binding domain for SST ligands is made up of residues in TMs III-VII with a potential contribution by the second extracellular loop. SSTRs are widely expressed in many tissues, frequently as multiple subtypes that coexist in the same cell. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated protein kinase (MAPK) through G-protein-dependent mechanisms. Some of the subtypes are also coupled to inward rectifying K(+) channels (SSTR2, 3, 4, 5), to voltage-dependent Ca(2+) channels (SSTR1, 2), a Na(+)/H(+) exchanger (SSTR1), AMPA/kainate glutamate channels (SSTR1, 2), phospholipase C (SSTR2, 5), and phospholipase A(2) (SSTR4). SSTRs block cell secretion by inhibiting intracellular cAMP and Ca(2+) and by a receptor-linked distal effect on exocytosis. Four of the receptors (SSTR1, 2, 4, and 5) induce cell cycle arrest via PTP-dependent modulation of MAPK, associated with induction of the retinoblastoma tumor suppressor protein and p21. In contrast, SSTR3 uniquely triggers PTP-dependent apoptosis accompanied by activation of p53 and the pro-apoptotic protein Bax. SSTR1, 2, 3, and 5 display acute desensitization of adenylyl cyclase coupling. Four of the subtypes (SSTR2, 3, 4, and 5) undergo rapid agonist-dependent endocytosis. SSTR1 fails to be internalized but is instead upregulated at the membrane in response to continued agonist exposure. Among the wide spectrum of SST effects, several biological responses have been identified that display absolute or relative subtype selectivity. These include GH secretion (SSTR2 and 5), insulin secretion (SSTR5), glucagon secretion (SSTR2), and immune responses (SSTR2).

Ligand-activated somatostatin receptors (SSTRs) initiate cytotoxic or cytostatic antiproliferative signals. We have previously shown that cytotoxicity leading to apoptosis was signaled solely via human (h) SSTR subtype 3, whereas the other four hSSTR subtypes initiated a cytostatic response that led to growth inhibition. In the present study we characterized the antiproliferative signaling mediated by hSSTR subtypes 1, 2, 4, and 5 in CHO-K1 cells. We report here that cytostatic signaling via these subtypes results in induction of the retinoblastoma protein Rb and G1 cell cycle arrest. Immunoblot analysis revealed an increase in hypophosphorylated form of Rb in agonist-treated cells. The relative efficacy of these receptors to initiate cytostatic signaling was hSSTR5hSSTR2hSSTR4 approximately = hSSTR1. Cytostatic signaling via hSSTR5 also induced a marginal increase in cyclin-dependent kinase inhibitor p21. hSSTR5-initiated cytostatic signaling was G protein dependent and protein tyrosine phosphatase (PTP) mediated. Octreotide treatment induced a translocation of cytosolic PTP to the membrane, whereas it did not stimulate PTP activity when added directly to the cell membranes. C-tail truncation mutants of hSSTR5 displayed progressive loss of antiproliferative signaling proportional to the length of deletion, as reflected by the marked decrease in the effects of octreotide on membrane translocation of cytosolic PTP, and induction of Rb and G1 arrest. These data demonstrate that the C-terminal domain of hSSTR5 is required for cytostatic signaling that is PTP dependent and leads to induction of hypophosphorylated Rb and G1 arrest.

The subset of striatal neurons which colocalize SS/NPY/NADPH-d are selectively resistant to neurodegeneration in Huntington's Disease (HD) and to excitotoxic cell death induced experimentally with NMDA receptor (NMDAR) agonists. Here we have analyzed the expression of immunoreactive NMDAR-1 (NR1) subunit (as an index of NMDAR protein) and of huntingtin (the normal product of the HD gene) in primary cultures of rat striatum to see if differential expression of the two antigens in the subset of SS/NPY/NADPH-d and other striatal neurons can explain their selective resistance or vulnerability. Double-label histochemical and immunocytochemical studies were carried out using conventional and confocal laser scanning microscopy to characterize the cellular and subcellular expression of NR1 and SS, or NPY or bNOS, together with NADPH-d histochemistry. The percentages of cultured striatal neurons that were positive for NADPH-d, SS, NPY, bNOS, and NRI were, respectively, 3.8, 8.4, 10.2, 5.1, and 80%. The majority of striatal NADPH-d neurons coexpressed SS and NPY; 17% of SS-producing neurons were strongly positive for NR1; the remaining cells (approximately 80%) exhibited only weak NR1 expression. Comparable data were obtained for NPY-positive neurons, 15% of which colocalized NR1 strongly and 70-80% weakly. By double-label immunofluorescence, huntingtin was nonselectively expressed in virtually all striatal neurons including SS/NPY/NADPH-d neurons. These results show that the majority of striatal SS/NPY/NADPH-d neurons express NR1. The relative abundance of NR1 in SS/NPY/NADPH-d neurons, however, varies between a small subset of neurons that are receptor rich and the remainder that express low levels only and may determine susceptibility to NMDAR-mediated neurotoxicity. Huntingtin is nonselectively expressed in virtually all striatal neurons and does not appear to be a determinant of the selective resistance of normal striatal SS/NPY/NADPH-d neurons to NMDA toxicity.

Somatostatin (SS) and neuropeptide Y (NPY) are coproduced in a subpopulation of neurons that are selectively resistant to NMDA neurotoxicity. We have previously reported that quinolinic acid (QUIN), an NMDA receptor agonist, augments SS mRNA in cultured fetal rat cortical neurons. This study examines coregulation of SS and NPY by QUIN and NMDA in cultured cortical neurons and compares the effects of these agents with those of forskolin and phorbol 12-myristate 13-acetate (PMA), known to activate SS and NPY gene transcription by protein kinase A- and protein kinase C-dependent mechanisms. In addition, transcriptional regulation of the SS gene was investigated by acute transfection of cortical cultures with an SS promoter-chloramphenicol acetyltransferase (CAT) construct. QUIN and NMDA displayed dose-dependent fourfold augmentation of levels of mRNA for SS but not for NPY. In contrast, forskolin and PMA increased both SS and NPY mRNA levels. QUIN- and NMDA-mediated induction of SS mRNA was blocked by the NMDA receptor antagonist (−)-2-amino-5-phosphonovaleric acid and displayed regional brain specificity because it was not observed in fetal hypothalamic cell cultures. In time course studies, the effects of QUIN/NMDA on SS mRNA occurred after a latency of 8 h, indicating a delayed effect. Cortical cells transfected with pSS-750 CAT showed three- to fourfold stimulation of CAT activity with forskolin but not by QUIN or NMDA. These data reveal a dose-dependent, tissue-specific, NMDA receptor-mediated stimulation of SS but not NPY mRNA. Such stimulation of SS gene expression does not require activation of the protein kinase A or protein kinase C signaling pathways, is not transcriptionally mediated, and is likely posttranscriptional, as also suggested by the delay in SS mRNA induction.

Using a cDNA probe, genomic clones were obtained for the 5′ flanking promoter sequence of the human somatostatin receptor 2-encoding gene (SSTR2). A 3.8-kb sequence directly upstream from the start codon was analyzed. This sequence shares a number of characteristics with the promoters of other G-protein-coupled receptor (GPCR)-encoding genes including a number of G + C-rich regions, binding sites for several transcriptional factors, and the absence of coupled TATAA and CAAT sequences.

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR 4 ). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst 1 , sst 2 , sst 3 , sst 4 and sst 5 ). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF 1 ) appears to comprise sst 2 , sst 3 and sst 5 receptor subtypes. The other class (SRIF 2 ) appears to comprise the other two recombinant receptor subtypes (sst 1 and sst 4 ). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression, and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR1-4 display weak selectivity for SST-14 binding whereas hSSTR5 is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3, and 5 belong to a similar SSTR subclass. hSSTR1 and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective, and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3-7 which is lined by hydrophobic and charged amino acid residues.

25-hydroxycholesterol (25-OHC), an oxysterol that potently regulates cellular cholesterol metabolism, induced formation of novel fibrillar structures in normal mouse astrocytes as observed by fluorescence microscopy with the cholesterol probe, filipin. These fibrils were identified as lysosomes by their immunoreactivity for the lysosome associated membrane glycoprotein (LAMP). In contrast, astrocytes derived from the Niemann-Pick disease type C (NPC) mutant mouse were resistant to this oxysterol-induced lysosomal reorganization. NPC astrocytes have abnormal intracellular cholesterol storage as observed by brightly positive filipin staining of their lysosomes. These results show that lysosomal cholesterol storage in NPC astrocytes is associated with a block in oxysterol-mediated fibrillar reorganization of lysosomes.

The somatostatin (SS) gene is transcriptionally regulated via the cyclic AMP (cAMP) response element (CRE), located in the proximal promoter (-41 to -48 bp). We have previously reported that glucocorticoids induce dose-dependent cell-specific alterations in the steady-state SS mRNA level. Here we have investigated direct transcriptional control of the SS gene by glucocorticoids. We have examined transcriptional interaction between glucocorticoids and the cAMP signalling pathway and mapped the 5′ upstream regulatory region of the SS gene involved in glucocorticoid transactivation. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in PC12 rat pheochromocytoma cells and A126-1B2 (protein kinase A-deficient mutant PC12) cells, by acute transfection of 5′ flanking SS DNA (- 750, -250 and -71 bp) ligated to the reporter (CAT) gene. Dexamethasone (DEX) induced a dose-dependent 2.2-fold stimulation of SS gene transcription in PC12 cells, but not in A126-1B2 cells. Other steroid and thyroid hormones tested, and retinoic acid, were ineffective, while cAMP and forskolin stimulated gene transcription 4-5-fold in PC12 cells but not in A126-1B2 cells. DEX exerted an additive effect on cAMP-induced gene transcription. Deletion of the promoter from -750 to -71 bp (but not from -750 to -250 bp) abolished all stimulatory effects of DEX without affecting cAMP responsiveness. Mutation of the CRE abrogated both DEX- and cAMP-dependent gene enhancement. Gel electrophoretic mobility shift assays confirmed that the -250 to -71 bp region of the SS promoter (but not the -71 to +55 bp domain) binds specifically to a glucocorticoid response element-sensitive nuclear protein(s) from PC12 cells, suggesting a putative glucocorticoid receptor interaction with SS promoter DNA. We conclude that glucocorticoids regulate SS gene transcription positively. Glucocorticoid-induced transactivation shows dependence on protein kinase. A activity, and may be mediated via protein-protein interaction between the glucocorticoid receptor and the CRE binding protein. DNA sequences upstream from the CRE between -250 and -71 bp in the SS promoter appear to be the target of glucocorticoid action.

We report here on the cloning of a human intronless gene encoding a member of the G-protein linked somatostatin (SST) receptor subfamily, termed SSTR3. Based on the deduced amino acid sequence, this gene encodes a 418 amino acid protein displaying sequence similarity, particularly within putative transmembrane domains, with the recently cloned human SSTR1 (62%), SSTR2 (64%) and SSTR4 (58%) receptors. Membranes prepared from COS-7 cells transiently expressing the human SSTR3 gene bound [125I]Leu8,D-Trp22,Tyr25 SST-28 in a saturable manner with high affinity (approximately 200 pM) and with rank order of potency (D-Trp8 SST-14SST-14SMS-201-995SST-28) indicative of a somatostatin-14 selective receptor. The pharmacological profile of the expressed human SSTR3 receptor is similar but not identical to that reported for the rat homolog [(1992) J. Biol. Chem. 267, 20422] where the peptide selectivity is SST-28or = SST-14SMS-201-995. Northern blot analysis reveals the presence of an SSTR3 mRNA species of approximately 5 kb in various regions of the monkey brain, including the frontal cortex, cerebellum, medulla, amygdala, with little or no SSTR3 mRNA detectable in brain regions such as the striatum, hippocampus, and olfactory tubercle. The SSTR3 receptor gene maps to human chromosome 22. The existence of at least four distinct human genes encoding somatostatin-14 selective receptors with diverse pharmacological specificities may help to account for some of the multiple biological actions of somatostatin under normal and pathological conditions.

Biological activity now recognizable as that of somatostatin (SS) was first encountered in 1968 by Krulich et al. [33] during attempts to screen hypothalamic extracts for growth hormone (GH) releasing activity. The workers identified a GH-inhibitory substance, characterized it as a low-molecular-weight basic peptide, and were able to localize the biological activity to the median eminence and anterior hypothalamic area. A year later, Hellman and Lernmark [23] reported the presence of a potent insulin-inhibitory factor in extracts of pigeon pancreatic islets. These two apparently unrelated observations were to come into sharp focus in 1973 with the discovery by Brazeau et al. [10] of the tetradecapeptide SS-14 as the hypothalamic GH-inhibitory substance, a signal achievement duly recognized by the award of the Nobel Prize to Guillemin in 1977. Subsequent studies revealed that SS is not produced only in the hypothalamus but also occurs throughout the central nervous system (CNS), in peripheral neurons, in the gastrointestinal (GI) tract and in the pancreatic islets of Langerhans. Furthermore, SS-like immunoreactivity is heterogeneous and is distributed in many tissues in both vertebrate and invertebrate species of the animal kingdom and in the plant kingdom. Extensive investigation of SS has shown that its wide anatomical distribution is paralleled by an equally broad spectrum of biological effects.

To determine whether somatostatin receptor subtypes arise from molecular heterogeneity of the receptor protein, we have cross-linked the putative receptor in normal rat tissues and in AtT-20 and GH3 cells, both chemically with SS-14, SS-28 and Tyr3 SMS ligands, as well as by photoaffinity labeling with an azido derivative of Tyr3 SMS (EE 581). Three prominent somatostatin receptor proteins of 58-kDa, 32-kDa, and 27-kDa size have been identified. These proteins exhibit a tissue-specific distribution, ligand selectivity, and relative preference for SS-14 and SS-28 binding, and thus qualify as somatostatin receptor subtypes. Using EE 581 as a photoaffinity probe, the 58-kDa and 32-kDa proteins have been purified to homogeneity from brain and AtT-20 cells by successive SDS-PAGE. The 58-kDa form has been trypsinized and amino acid sequence data obtained from four tryptic fragments. With the help of synthetic oligonucleotides derived from these sequences, work is currently in progress to clone the 58-kDa protein to elucidate its complete sequence, its expression, and its functional relationship to the somatostatin receptor and its pharmacological subtypes.

Adrenocortical somatostatin receptors have been shown to interact with somatostatin-14 (S-14) and somatostatin-28 (S-28). To determine whether these peptides interact with the same or different receptor proteins, we chemically cross-linked these receptors using disuccinimidyl suberate to radioligands prepared from tyrosinated S-14 and S-28 analogs. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and subsequent autoradiography of [125I-Tyr11] S-14 and [Leu8, D-Trp22, 125I-Tyr25] S-28 cross-linked to their binding sites following solubilization in the presence of 50 mM DTT revealed the presence of a single labelled protein of Mr = 200,000. When the cross-linked material was treated under non-reducing conditions, this band was not observed. Furthermore, addition of excess S-14 and S-28 at the time of binding inhibited the incorporation of both radioligands into the receptor protein. These results demonstrate that adrenocortical membrane receptors for somatostatin contain a single receptor protein sub-unit or sub-units of identical size which interact with both S-14 and S-28.

Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14§) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28§) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14§ (with or without unlabeled S-14) or 17 pmol S-28§ (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14§- and S-28§-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14§ and S-28§ (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14§ was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina ter...

To determine directly whether somatostatin-28 (S-28) physiologically regulates GH secretion, and what its contribution is relative to somatostatin-14 (S-14), we have compared the effects of immunoneutralization with a specific S-28 antibody with those of an anti S-14/S-28 serum on GH secretory dynamics in the rat. Plasma samples were obtained every 15 min for 7 h (1000-1700 h) from conscious, chronically cannulated rats after iv administration of 1 ml of one of the following sera: 1) rabbit anti S-28 (reacts with S-28, but not with S-14; maximum binding, 297 pmol/ml), 2) nonimmune rabbit serum, 3) sheep anti-S-14/S-28 serum (maximum binding, 9.4 nmol S-14 or S-28/ml), and 4) nonimmune sheep serum. A comparison of the mean integrated plasma GH levels during peak and trough periods showed significantly higher trough GH levels in both antibody-treated groups compared to those in the corresponding controls. In the anti-S-14/S-28-treated group, the elevation of trough period GH levels (40.5 +/- 3.5 ng/ml) represented a 3.25-fold increase (P less than 0.01) compared to the control value (12.5 +/- 1.5 ng/ml). In the anti-S-28-treated group, trough period GH levels (14 +/- 1.6 ng/ml) showed a 2.3-fold increase (P less than 0.01) compared to the control value (6.1 +/- 0.9 ng/ml). Mean peak period GH levels were 1.35-fold higher (P less than 0.05) than control values in the anti-S-14/S-28-treated group; anti-S-28 serum did not change mean peak GH levels. These data provide strong evidence that circulating S-28 (like S-14) physiologically regulates trough GH secretion and that the contribution of S-28 to GH inhibition is as important as that of S-14.

We examined the effects of intracerebroventricular (icv) administration of the somatostatin peptides, S-14 and S-28, and an analog, [D-Trp22]S-28, on spontaneous GH and glucose secretion in freely moving rats bearing chronic icv and intraatrial cannulae. Normal saline icv-treated control rats exhibited the typical pulsatile pattern of GH secretion. Central injection of S-14, S-28, and [D-Trp22]S-28 (each at two different doses, 5 and 10 micrograms) caused an early significant suppression of plasma GH levels which was of longer duration after S-28 or [D-Trp22]S-28 than after S-14 injection. The icv administration of S-28 and [D-Trp22]S-28 (but not S-14) also resulted in significant hyperglycemia, which persisted for periods up to 1 h. In a second study designed to determine whether somatostatin administered via the brain ventricle can reach the peripheral circulation, we measured plasma levels of S-14-like immunoreactivity (S-14 LI) and S-28-(15-28) LI at frequent time intervals after icv injection of S-14 (5 micrograms) and S-28 (10 micrograms). Plasma S-14 LI rose from a basal value of 0.16 +/- 0.02 (+/- SE) ng/ml to a peak of 3.0 +/- 1.0 ng/ml at 1 min, and S-28 icv resulted in a 150-fold increase in plasma S-28-(15-28) LI 1 min after injection. Sephadex G-50 gel chromatography revealed that the plasma immunoreactivity consisted of a single molecular species (either S-14 or S-28) corresponding to the centrally administered counterpart. These results demonstrate that after icv administration at high doses, both forms of somatostatin cause an acute inhibition of spontaneous GH secretion and that the larger form also causes hyperglycemia. We correlate these events with massive hypersomatostatinemia resulting from leakage of the somatostatin peptides from the cerebrospinal fluid into the systemic circulation. The findings indicate that icv injected hypothalamic hormones can reach the peripheral circulation and exert significant biological actions on distant target organs; thus, they have important implications for the design and interpretation of experiments in which peptides are administered via the cerebral ventricles.