Somatostatin receptors (sst2) regulate cGMP production in rat retina

The present study investigated the effect of somatostatin in the regulation of cGMP levels in rat retina and the mechanisms involved in this process. Isolated rat retinas were treated alone or in the presence of somatostatin (0.01-10 microM), BIM23014 (sst2 agonist, 0.01-10 microM), L-796,778 (sst3 agonist, 10 microM), somatostatin (0.1 microM) in combination with CYN154806 (sst2 antagonist, 1 microM), N(G)-methyl-L-arginine acetate salt (NMMA, inhibitor of the nitric oxide synthase (NOS), 250 microM), orthovanadate (inhibitor of tyrosine phosphatase, SHP-1, 1 microM), and arginine alone (250 microM). cGMP levels were quantified by ELISA. Immunohistochemistry studies were performed for the detection of cGMP and nNOS, while Western blot analysis was employed for the detection of SHP-1. Somatostatin increased cGMP levels in a concentration-dependent manner. This increase was inhibited by CYN154806. BIM23014 increased cGMP levels only at the concentration of 10 microM, while L-796,778 had no effect. NMMA blocked completely the somatostatin stimulated increase of cGMP levels and nNOS was detected in rat retina. cGMP immunoreactivity was observed primarily in bipolar cells only of nitroprusside-treated retinas. SHP-1 inhibition by orthovanadate reduced the somatostatin effect in a statistically significant manner. These results suggest that a SRIF/SHP-1/NO/cGMP mechanism underlies the actions of somatostatin in the retina and in its influence of retinal circuitry.     

Somatostatin receptors (sst2) are coupled to Go and modulate GTPase activity in the rabbit retina

The role of somatostatin and its mechanism of action in the retina remains an important target for investigation. Biochemical and pharmacological studies were engaged to characterize the somatostatin receptors in the rabbit retina, and their coupling to G-proteins. The ability of selective ligands to inhibit [125I]Tyr11-somatostatin-14 binding to rabbit retinal membranes was examined. The sst2 analogues SMS201-995, MK678, and BIM23014, displayed IC50 values of 0.28 +/- 0.12, 0.04 +/- 0.01 and 1.57 +/- 0.39 nm, respectively. The sst1 analogue CH275 moderately displaced the [125I]Tyr11-somatostatin-14 binding, while selective analogues for sst3, sst4 and sst5 had minimal effect. Immunoblotting and/or immunohistochemistry studies revealed the presence of the pertussis toxin sensitive Gi1/2, and Go proteins, as well as Gs. Somatostatin-14 and MK678 stimulated GTPase activity in a concentration-dependent manner with EC50 values of 42.8 +/- 16.8 and 70.0 +/- 16.5 nm, respectively, thus supporting the functional coupling between the receptor and the G-proteins. CH275 stimulated the GTPase activity moderately, in agreement with its binding profile. The antisera raised against Goalpha and Gi1/2alpha inhibited the somatostatin-induced high-affinity GTPase activity, but only anti-Goalpha inhibited the MK678 stimulation of the enzyme. These results suggest that somatostatin mediates its actions in the rabbit retina by interacting mainly with sst2 receptors that couple to Goalpha.     

Nonpeptidyl somatostatin agonists demonstrate that sst2 and sst5 inhibit stimulated growth hormone secretion from rat anterior pituitary cells.

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P < 0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.     

Immunohistochemical Distribution and Subcellular Localization of the Somatostatin Receptor Subtype 1 (sst1) in the Rat Hypothalamus

The aim of the present study was to examine the cellular and sub-cellular distribution of the somatostatin (SRIF) receptor subtype sst1 in the rat hypothalamus. Receptors were immunolabeled using an antibody directed against an antigenic sequence in the N-terminus of the receptor. Immunopositive neuronal cell bodies and dendrites were observed throughout the mediobasal hypothalamus, including the medial preoptic area, paraventricular, periventricular, and arcuate nuclei. Immunoreactive axons and axon terminals were also observed in the median eminence, suggesting that sst1 is also located pre-synaptically. Electron microscopic examination of the arcuate nucleus revealed a predominant association of immunoreactive sst1 with perikarya and dendrites. Most immunoreactive receptors were intracellular and localized to tubulovesicular compartments and organelles such as the Golgi apparatus, but 14% were associated with the plasma membrane. Of the latter, 47% were apposed to abbuting afferent axon terminals and 20% localized immediately adjacent to an active synaptic zone. These results demonstrate a widespread distribution of sst1 receptors in rat hypothalamus. They also show that somatodendritic sst1 receptors in the arcuate nucleus are ideally poised to mediate SRIF’s modulation of afferent synaptic inputs, including central SRIF effects on growth hormone-releasing hormone neurons documented in this area.Special Issue Dedicated to Miklós Palkovits.     

Genistein stimulates electrogenic Cl(-) secretion in mouse jejunum

We used the short-circuit current (I_sc) technique to investigate the effects of the isoflavone genistein on the electrogenic Cl^– secretion of the mouse jejunum. Genistein stimulated a sustained increase in I_sc that was dose dependent. Bumetanide inhibited 76 ± 5% of the genistein-stimulated I_sc consistent with activation of Cl^– secretion. Genistein failed to stimulate I_sc following maximal activation of the cAMP pathway by forskolin. In addition, forskolin had a reduced effect on I_sc of the mouse jejunum in the presence of genistein. Glibenclamide, a blocker of CFTR, eliminated the genistein-stimulated increase of I_sc and reduced the forskolin-activated I_sc. Clotrimazole, a Ca²⁺-activated K⁺ channel blocker, failed to reduce the genistein-stimulated I_sc. Vanadate, a blocker of tyrosine-dependent phosphatases, reduced the genistein-activated I_sc. Tyrphostin A23, a tyrosine kinase inhibitor, reduced basal I_sc, after which genistein failed to stimulate I_sc. These data suggest that genistein activated a sustained Cl^– secretory response of the mouse jejunum and that the effect of genistein was via a tyrosine-dependent phosphorylation pathway. 1-ethyl-2-benzimidazolone; vanadate; tyrphostin A23; cantharidic acid; phosphatase     

The effect of selective sst1, sst2, sst5 somatostatin receptors agonists, a somatostatin/dopamine (SST/DA) chimera and bromocriptine on the "clinically non-functioning" pituitary adenomas in vitro

The aim of the work was to investigate the effects of somatostatin analogs acting selectively on sst1 (BIM-23926), sst2 (BIM-23120) and sst5 (BIM-23206) receptor subtypes on the viability of "clinically non-functioning" pituitary adenomas in vitro. The effects of native SST (SST-14), a SST/DA chimera (BIM-23A387) and a D(2)-dopamine receptor agonist bromocriptine (BC) were also examined. The study was performed on 10 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". A part of each tumor was mechanically dispersed and digested with collagenase to isolate the tumoral cells. Another part of each tumor was fixed, embedded in paraffin and immunostained to reveal the pituitary hormones and SST receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5). The tumoral cell suspensions were incubated for 24 h with the substances mentioned above. The quantity of viable cells was estimated using the EZ4U system. The results were compared with the immunohistochemical evaluation of the hormonal profile of adenoma and the sst receptor subtype immunoreactivities present. The findings indicate that selective sst1, sst2 and sst5 receptors agonists, SST/DA chimera and D(2)-dopamine receptor agonist bromocriptine affect the viability of some, but not all, "clinically non-functioning" pituitary adenomas in vitro. The most effective was bromocriptine. The investigated somatostatin analogs including SST/DA chimera exerted roughly similar inhibitory effects. Further studies are needed to fully evaluate the potential usefulness of these compounds in the pharmacological treatment of "non-functioning" pituitary tumors.     

Protein synthesis and secretion by hamster, rat and mouse jejunum

The synthesis and secretion of [35S]methionine-labelled proteins by hamster, rat and mouse jejunum has been measured in vitro after 60 min incubation in culture medium. Twenty-three major bands of radioactively labelled proteins were detected by PAGE analysis of enterocyte extracts. Three of these proteins having mol. wts of 28,000, 43,000 and 60,000 appeared to be synthesised preferentially by older enterocytes. A small number of radioactively labelled proteins also appeared in culture medium at the end of incubation. Three of these proteins, accounting for most of the recovered radioactivity, had mol. wts of 28,000, 43,000 and 60,000. Secretion of these proteins was inhibited by monensin. Further experiments showed these secreted proteins to be acidic and possibly glycoprotein in nature. Their rapid turnover, selective secretion and changing abundance in enterocytes of different ages all suggest that they may have important functions to perform in the intestine.     

Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A)

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst(2A) and sst(3) exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and sst(3). The sst(2A)-sst(3) heterodimer exhibited high affinity binding to somatostatin-14 and the sst(2)-selective ligand L-779,976 but not to the sst(3)-selective ligand L-796,778. Like the sst(2A) homodimer, the sst(2A)-sst(3) heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst(2)-selective ligand L-779,976. However, unlike the sst(3) homodimer, the sst(2A)-sst(3) heterodimer did not promote GTPgammaS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst(3)-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst(2A)-sst(3) heterodimer desensitized at a slower rate than the sst(2A) and sst(3) homodimers. Both sst(2A) and sst(3) homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst(2A)-sst(3) heterodimer separated at the plasma membrane, and only sst(2A) but not sst(3) underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst(2A) and sst(3) results in a new receptor with a pharmacological and functional profile resembling that of the sst(2A) receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst(3) receptor function by heterodimerization with sst(2A) or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst(3)-specific binding and signaling in mammalian tissues.     

Somatostatin inhibits follicle-stimulating hormone-induced adenylyl cyclase activity and proliferation in immature porcine Sertoli cell via sst2 receptor

The potential involvement of somatostatin (SRIF) in testicular function was studied by using as a model primary cultures of purified immature porcine Sertoli cells. In the present report we show that Sertoli cells express mRNA for sst2 SRIF receptor and display SRIF-sensitive adenylyl cyclase. Sensitivity of adenylyl cyclase to SRIF and its analogues is compatible with the pharmacological profile of this receptor type. Relevant cAMP production is similarly inhibited by SRIF in both basal and stimulated (by gonadotropin FSH or by forskolin) conditions. Moreover, the observed SRIF actions on Sertoli cells require functional coupling of specific membrane receptors to adenylyl cyclase via Gi proteins because pertussis toxin prevents SRIF-dependent inhibition of adenylyl cyclase in either basal or FSH-stimulated conditions. Given the potent antiproliferative actions of SRIF in other cell types, we further assessed the possible SRIF-dependent modulation of [(3)H]thymidine incorporation by Sertoli cells. Our data point to SRIF-mediated inhibition of both basal and FSH-stimulated [(3)H]thymidine uptake. This inhibition of Sertoli cell proliferation is, at least in basal conditions, also blocked by pertussis toxin pretreatment. Altogether, these data suggest that SRIF may play a role as an (local) inhibitor of FSH actions in testicular development.     

Somatostatin receptors in brain and pituitary

Somatostatin (SRIF) actions in the brain and pituitary are mediated by specific receptors. Using radioiodinated ligands it has been possible to characterize the kinetics of specific binding sites in the brain and pituitary, and to determine their cellular localization by autoradiography. At the pituitary level, the inhibition of growth hormone, prolactin and thyrotropin secretions induced by SRIF is mediated through a single binding site which is coupled to the inhibition of adenylate cyclase. In the brain, SRIF receptors are localized on neurons and glial cells and are also coupled to adenylate cyclase inhibition. Two sites are differentiated in the brain with an analogue of somatostatin, SMS 201995. In humans, SRIF-binding sites have been related to a number of pathologies. At the pituitary level, it has been shown that the number of binding sites was negatively correlated to growth hormone levels in acromegaly. Furthermore, SRIF-binding sites were undetectable in a patient which did not respond to SMS 201995 therapy. In the brain, meningiomas and gliomas are rich in SRIF binding sites. This suggests a possible role for SRIF on glia. In neurodegenerative diseases, cortical SRIF concentrations are decreased in Alzheimer's and Parkinson's disease associated with dementia while SRIF-binding sites are only affected in Alzheimer's disease. In conclusion, the physiological role of SRIF in the brain and pituitary can be evaluated by studying the receptors of the peptide. Such studies allow to question the implication of SRIF in endocrine and neuropathologies.     

Somatostatin receptors in the senescent rat brain: a quantitative autoradiographic study.

To examine the effects of aging on the density and distribution of somatostatin receptors (SS-R) in the rat brain, receptor autoradiography for SS-R was carried out in rats aged 3 and 24 months using 125I-labeled Tyr11-SS-14. Autoradiograms were quantitatively assessed by an image analyzer to evaluate changes in the expression of SS-R due to senescence. Statistically significant decreases in SS-R binding were found in specific regions of the brains of senescent rats as compared to young adult rats. The regions affected included the periaqueductal gray matter (73% loss versus young adult rats), the interpeduncular nucleus (73% loss), the pontine nucleus (63% loss), the superior colliculus (46% loss), the ventral tegmental area (46% loss), the temporal cortex (39% loss), the frontal cortex (34% loss), the hippocampus (33% loss), the amygdala (27% loss) and the claustrum (26% loss). There was no significant change in SS-R expression in the spinal cord with aging. Significant reductions in SS-R binding in these brain regions may be involved in the impairment of sensory and cognitive function that can occur with aging.     

sst2 Somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.     

Adenosine A2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons

Summary Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP--S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.     

The Truncated Isoform of Somatostatin Receptor5 (sst5TMD4) Is Associated with Poorly Differentiated Thyroid Cancer

Somatostatin receptors (ssts) are expressed in thyroid cancer cells, but their biological significance is not well understood. The aim of this study was to assess ssts in well differentiated (WDTC) and poorly differentiated thyroid cancer (PDTC) by means of imaging and molecular tools and its relationship with the efficacy of somatostatin analog treatment. Thirty-nine cases of thyroid carcinoma were evaluated (20 PDTC and 19 WDTC). Depreotide scintigraphy and mRNA levels of sst-subtypes, including the truncated variant sst5TMD4, were carried out. Depreotide scans were positive in the recurrent tumor in the neck in 6 of 11 (54%) PDTC, and in those with lung metastases in 5/11 cases (45.4%); sst5TMD4 was present in 18/20 (90%) of PDTC, being the most densely expressed sst-subtype, with a 20-fold increase in relation to sst2. In WDTC, sst2 was the most represented, while sst5TMD4 was not found; sst2 was significantly increased in PDTC in comparison to WDTC. Five depreotide positive PDTC received octreotide for 3–6 months in a pilot study with no changes in the size of the lesions in 3 of them, and a significant increase in the pulmonary and cervical lesions in the other 2. All PDTC patients treated with octreotide showed high expression of sst5TMD4. ROC curve analysis demonstrated that only sst5TMD4 discriminates between PDTC and WDTC. We conclude that sst5TMD4 is overexpressed in PDTC and may be involved in the lack of response to somatostatin analogue treatment.     

Expression and function of KCNH2 (HERG) in the human jejunum

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERG(USO)) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3-S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10(-6) M), atropine (10(-6) M). and l-nitroarginine (10(-4) M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10(-8) M) and MK-499 (3 x 10(-8) M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10(-6) M) produced a 10-20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10(-6) M) did not affect [(14)C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.     

Role of liver-type glucose transporter (GLUT2) in transport across the basolateral membrane in rat jejunum.

To obtain information on the regulation of glucose transport across the basolateral membrane (BLM) of intestinal epithelial cells, we measured the number of [3H]cytochalasin B binding sites and the level of liver-type glucose transporter (GLUT2) protein in the BLM in the jejunum of rats (i) with diabetes (ii) given a high-carbohydrate diet or (iii) with experimental hyperglycemia (12 h infusion of a high-glucose solution). A glucose uptake and the number of D-glucose inhibitable [3H]cytochalasin B binding sites in BLM vesicles were significantly increased in all three conditions. Western blot analysis showed that the amount of GLUT2 protein in BLM vesicles was increased in rats with diabetes and those given a high-carbohydrate diet, but not in those with experimental hyperglycemia. These results suggest that there is a mechanism for rapid regulation of glucose transport in the BLM that does not depend on change in the amount of GLUT2.     

Somatostatin increases mitogen-induced IL-2 secretion and proliferation of human jurkat T cells via sst3 receptor isotype

The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (K_I1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using ¹²⁵I-Tyr¹-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC₅₀ = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC₅₀ concentrations for both actions were almost identical (EC₅₀ = 22 ± 9 pM and EC₅₀ = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62–73, 1998. © 1998 Wiley-Liss, Inc.     

Structural determinants of agonist-selective signaling at the sst(2A) somatostatin receptor

The clinically used somatostatin (SS-14) analogs octreotide and pasireotide (SOM230) stimulate distinct species-specific patterns of sst(2A) somatostatin receptor phosphorylation and internalization. Like SS-14, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues, namely S341, S343, T353, T354, T356, and T359, which in turn leads to a robust endocytosis of both rat and human sst(2A) receptors. Unlike SS-14, pasireotide fails to induce any substantial phosphorylation or internalization of the rat sst(2A) receptor. Nevertheless, pasireotide is able to stimulate a selective phosphorylation of S341 and S343 of the human sst(2A) receptor followed by a clearly detectable receptor sequestration. Here, we show that transplantation of amino acids 1-180 of the human sst(2A) receptor to the rat sst(2A) receptor facilitates pasireotide-induced internalization. Conversely, construction of a rat-human sst(2A) chimera conferred resistance to pasireotide-induced internalization. We then created a series of site-directed mutants leading to the identification of amino acids 27, 30, 163, and 164 that when exchanged to their human counterparts facilitated pasireotide-driven S341/S343 phosphorylation and internalization of the rat sst(2A) receptor. Exchange of these amino acids to their rat counterparts completely blocked the pasireotide-mediated internalization of the human sst(2A) receptor. Notably, octreotide and SS-14 stimulated a full phosphorylation and internalization of all mutant sst(2A) receptors tested. Together, these findings suggest that pasireotide activates the sst(2A) receptor via a molecular switch that is structurally and functionally distinct from that turned on during octreotide-driven sst(2A) activation.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.