Specific Binding Sites For Somatostatin Research Articles

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 (K11 = 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.

Distribution and pharmacological characterization of somatostatin receptor binding sites in the sheep brain

Somatostatin binding sites have been localized and quantified in the sheep brain using 125I-Tyr0-DTrp8-somatostatin, by quantitative high resolution light microscopic autoradiography. Sections were analyzed by densitometry on radioautographic film, and subsequently on slides coated with photoemulsion. Specific somatostatin binding sites were concentrated in the medial habenula, superior colliculus, dorsal motor nucleus of the vagus nerve, inferior olive, spinal trigeminal nucleus, and cerebellum. In competition experiments, octreotide, a sst2/sst3/sst5 selective agonist only partially displaced 125I-Tyr0-DTrp8-somatostatin in the three cerebellar layers while it was fully active as compared to somatostatin 14 and 28 in the deeper layers of the parietal cortex. Moderate to low somatostatin receptor densities were present in the mesencephalic periaqueductal gray, dorsal raphe, thalamic paraventricular nucleus, interpeduncular nucleus, pineal gland, dorsal tegmental, dorsolateral tegmental and parabrachial nuclei, nucleus of the solitary tract. The distribution of somatostatin binding sites generally correlates with the data obtained on slides dipped in photoemulsion which provided better resolution and more precise localization. In most of the labeled areas, 125I-Tyr0-DTrp8-somatostatin receptor binding was distributed between both neuropil and perikarya. Perikarya bearing 125I-Tyr0-DTrp8-somatostatin receptors were observed in areas which did not display detectable binding sites on film such as the preoptic-anterior hypothalamic complex and arcuate nucleus and in the locus coeruleus. In conclusion, the distribution of 125I-Tyr0-DTrp8-somatostatin binding sites in sheep brain is very reminiscent of other mammals being closer to the human than to rodents.

Inhibitory effect of a somatostatin analogue (SMS 201-995) on the growth of androgen-dependent mouse mammary tumor (Shionogi carcinoma 115).

The influence of a soinatostatin analogue, SMS 201–995 (SMS), on the growth of an androgen‐dependent mouse mammary tumor, Shionogi carcinoma 115 (SC115), was studied. Treatment of SC11S tumor‐transplanted male mice with s.c. injections of SMS (0.04, 0.2,1, and 5 μg twice a day) resulted in a dose‐dependent inhibition of tumor growth. The growth‐inhibitory effect of SMS reached its peak at a dose of 1 μg twice a day. SMS was found not to elicit its growth‐inhibitory effect through lowering plasma testosterone levels or down‐regulating androgen receptor of SC115 tumors. Since specific binding sites for somatostatin were not observed in the membrane fractions of SC115 tumors and SMS did not inhibit the proliferation of primarily cultured SC115 tumor cells, a direct inhibitory mechanism of SMS on SC115 tumors was unlikely to be operative. Since SMS is a very potent inhibitor of growth hormone (GH) secretion, it was speculated that SMS might inhibit the growth of SC115 tumors indirectly through down‐regulation of plasma GH levels. This possibility was evaluated by studying the influence of GH replacement on the growth of SC115 tumors grown in SMS‐treated mice. GH replacement was done both in a male secretory pattern (intermittent injection, human GH 500 μg/kg twice a day) and in a female secretory pattern (continuous infusion, 1000 μg/kg/day). Intermittent injections of GH fully restored the growth of SC115 tumors in the SMS‐treated mice to that in the normal controls but continuous infusion of GH was without effect. These results suggest that SMS inhibits the growth of SC115 tumors through suppression of GH secretion, and that the mode of GH administration is an important determinant of its action on SC115 tumor growth.

The binding of bombesin and somatostatin and their analogs to human colon cancers.

Specific receptors for bombesin/gastrin-releasing peptide, somatostatin, and EGF were investigated in 15 human colon cancer specimens. Eight of 15 clinical specimens (15%) of colon cancer showed the presence of somatostatin receptors. Octapeptide somatostatin analogs, RC-160 and RC-121, showed 10 times higher binding affinity for somatostatin receptors on colon cancer membranes than somatostatin. Analysis of 125I-Tyr4-bombesin binding data revealed the presence of specific binding sites in six (40%) specimens of human colon cancer. Scatchard analysis of 125I-labeled bombesin indicated a single class of receptors in three specimens with an apparent Kd value of 2.5 nM and two classes of receptors with high (Kd = 0.4 +/- 0.2 nM) and low affinity (Kd = 1.6 +/- 0.4 microM) in three other specimens. The 125I-Tyr4-bombesin binding capacities in the colon cancers for high affinity binding sites were from 6 to 228 fmol/mg protein and for low affinity binding sites 76 +/- 15 pmol/mg protein. None of the membrane preparations made from normal colonic mucosa specimens showed specific binding for 125I-Tyr4-bombesin. Five pseudononapeptide (psi 13-14) bombesin (6-14) antagonists, with different modifications at Positions 6 and 14, synthesized in our laboratory, inhibited the binding of 125I-Tyr4-bombesin in nanomolar concentrations. No correlation was found between the degree of differentiation and the presence of binding sites for somatostatin or bombesin. Specific binding of EGF was detected in 80% of colon cancer specimens. EGF binding capacity in colon cancer membranes was on average twice as high as in normal colon mucosa (50 +/- 21 vs 28 +/- 14 fmol/mg protein, respectively). Specific binding sites for somatostatin and EGF, but not bombesin, were also demonstrated in human colon cancer cell line HT-29. In HCT-116 colon cancer line only EGF receptors were found. These receptor findings and our in vivo studies on inhibition of colon cancer growth support the merit of continued evaluation of somatostatin analogs and bombesin/gastrin-releasing peptide antagonists in the management of colonic carcinoma.

Somatostatin receptors are restricted to a subpopulation of osteoblast-like cells during endochondral bone formation

Specific binding sites for the peptide hormone somatostatin have previously been demonstrated in long bones from neonatal rats. In the present study, the distribution of somatostatin receptors during embryonic bone formation has been investigated using the stable radioiodinated somatostatin analogue, SDZ 204-090. Somatostatin receptors in rat long bones were first detectable at the time of invasion of the cartilage model by osteogenic cells. Initially, receptors were detectable throughout the region occupied by osteogenic cells. As bone growth proceeded, however, receptors were restricted to the region of most recent invasion of the hypertrophic cartilage, where osteoid had not yet been deposited. In vivo labelling studies in neonatal rats were carried out to identify the cells bearing somatostatin receptors. Receptors were present in a restricted region of the metaphysis, immediately adjacent to the hypertrophic cartilage. Chondrocytes, osteoclasts, and mature osteoblasts were not labelled by the radioligand. The labelled cells were often apposed to remnants of cartilage matrix and stained positively for the osteoblast marker, alkaline phosphatase. Thus the cells with specific somatostatin-binding sites were probably osteoblast precursor cells. Specific binding was detectable in all endochondral bones examined, including those of the skull, but no specific binding was found in the membrane bones of the skull. These data suggest that somatostatin is involved in the regulation of osteoblast differentiation during endochondral bone formation.

Specific somatostatin binding sites in bone cells of newborn rats identified by [125I]204-090

Specific somatostatin binding sites in bone cells of newborn rats identified by [125I]204-090

Effects of alloxan-induced diabetes on somatostatin binding to cytosolic components of rabbit gastric fundic mucosa.

Diabetes was induced by administration of alloxan (150 mg/Kg) to 24 h-fasted rabbits. Somatostatin-like immunoreactivity (SLI) and cytosolic binding sites for somatostatin in gastric fundic mucosa were studied using radiolabelled Tyr11-somatostatin. Three months after the onset of the disease, the specific binding of somatostatin to these sites was seen to be significantly lower, due to a reduction in the number (but not the affinity) of specific somatostatin binding sites of high-affinity and a disappearance of the specific somatostatin binding sites of low-affinity. These changes were associated with an increase in the SLI concentration in both gastric fundic mucosa and plasma.

Effects of fasting and refeeding on somatostatin concentration and binding to cytosol from rabbit gastric mucosa.

Somatostatin like immunoreactivity and the density of somatostatin binding sites were measured in stomach (fundus and antrum) from either fed, 12 to 96 hours fasted, or 96 hours fasted plus 48 hours refed rabbits. The somatostatin concentration increased in fundic and antral mucosa after 24 h and reached its highest value after 96 h of fasting. The number of specific somatostatin binding sites with high and low affinity decreased with the duration of fasting. Refeeding of fasted animals resulted in a normalisation to control values of gastric mucosal somatostatin and somatostatin binding.

Characterization of specific somatostatin binding sites in guinea pig lung

Somatostatin binding sites have been demonstrated in the cytosolic fraction of guinea-pig lung. Binding of 125I-Tyr 11-somatostatin was dependent on time and temperature, saturable, reversible and highly specific. Under equilibrium condition, i.e. 60 min at 25°C, native somatostatin inhibited tracer binding in a dose-dependent manner. Two types of somatostatin binding sites were defined by Scatchard analysis: a small population with a high affinity ( K d = 23.4 nM) and a large population with a low affinity ( K d = 253.5 nM) for somatostatin. The biphasic nature of the dissociation process confirmed the heterogeneity of somatostatin binding sites. Apart from somatostatin, no peptide (1 μM) tested influenced the binding of 125I-Tyr 11-somatostatin. The present data represent the first analysis of somatostatin binding sites in lung.

Postnatal development of somatostatin levels and its binding sites in rabbit gastric mucosa

Using a specific radioimmunoassay technique, we have determined somatostatin-like immunoreactivity (SLI) in acid extracts of gastric (fundic and antral) mucosa as well as the specific binding of 125I-Tyr 11-somatostatin to cytosol of the stomach of 0 to 150 days postnatal rabbits. The levels of somatostatin in both fundus and antrum decreased from birth up to day 5 followed by a sharp increase from 5 to 10 days, then decreased progressively until day 35. After this age, the somatostatin concentration remained relatively stable. The number of specific somatostatin binding sites of both high- and low-affinity increased gradually (without changes in the affinity values) with the development of rabbits, reaching the adult level by 35 days. However, there was an apparent lack of high-affinity sites immediately after birth (day 0). The somatostatin binding sites had characteristics identical with those found in adult animals with regard to their respective specific ligands.

Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini.

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

Identification and characterization of specific binding sites for somatostatin in cytosol of bovine cystic duct mucosa.

Specific binding sites for somatostatin have been detected in cytosolic fraction of bovine cystic duct mucosa. At 37 degrees C, the interaction of 125I-Tyr11-somatostatin with cytosolic fraction was rapid, reversible, specific and saturable. At equilibrium, the binding of tracer was competitively inhibited by native peptide in the 1 nM to 2 microM range of concentrations. Scatchard analysis of binding data suggested the presence of two distinct classes of somatostatin binding sites: a class with a high affinity (Kd = 7.8 +/- 0.3 nM) and a low capacity (1.3 +/- 0.3 pmol somatostatin/mg protein) and a class with a low affinity (Kd = 129.1 +/- 2.0 nM) and a high capacity (43.5 +/- 6.7 pmol somatostatin/mg protein). The binding sites were shown to be highly specific for somatostatin since neuropeptides present in cystic duct such as Leu-enkephalin, neurotensin, substance P and vasoactive intestinal peptide did practically not show competition. These findings suggest that somatostatin could contribute to the regulation of the functions of the cystic duct mucosa in physiological and pathological conditions.

Somatostatin binding sites in human leptomeninx

Specific binding sites for somatostatin (SRIF) have been visualized in the human leptomeninx by means of autoradiographical techniques using the stable SRIF octapeptide analog 125I-204-090 as radioligand. The binding sites are specific for SRIF since only biologically active SRIF analogs compete with the radioligand. The density of the binding sites is comparable to that of the adjacent cortical structure. The presence of SRIF binding sites in the human leptomeninx, together with the recent demonstration of SRIF binding sites in human meningiomas, strongly suggest that this peptide may play a role in the physiology of the leptomeninx.

Interaction of somatostatin with isolated cytosol from rabbit renal papilla

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rabbit renal papilla. The interaction of 125I-Tyr 11-somatostatin with cytosolic fraction was rapid, reversible, specific, saturable and dependent on temperature. At 25°C the binding data were compatible with the existence of two classes of binding sites: a high-affinity class with a K d =57.7 nM and a low-affinity class with a K d =217.4 nM . Somatostatin binding sites exhibited a high degree of specificity since neuropeptides such as Leu-enkephalin, neurotensin, substance P, vasopressin and vasoactive intestinal peptide behaved as ligands with null or very low affinity.

Characterization of somatostatin binding sites in cytosolic fraction of pig retina

Specific binding sites for somatostatin have been characterized in the cytosolic fraction of the pig retina. The interaction of 125I-Tyr 11-somatostatin with cytosolic fraction was rapid, reversible, specific, saturable and temperature-dependent. At 15°C, the binding data were compatible with the existence of two classes of binding sites: a high-affinity class with a K d of 16.1 nM and a low-affinity class with a K d of 236.4 nM. The presence of somatostatin binding sites in the cytosolic fraction of the retina, together with the known occurrence of somatostatin at this level, strongly suggest that this peptide may be involved in the physiology of the retina.

Evidence for somatostatin binding sites in rabbit kidney

Specific binding sites for somatostatin have been identified in cytosolic fraction of rabbit kidney (cortex and outer medulla) using 125I-Tyr 11-somatostatin. The binding was saturable and reversible, as well as time and temperature dependent. Optimal pH for binding was observed at about 7.4. Scatchard plots were compatible with the existence of two classes of binding sites: a first class with a high affinity ( K d = 40 nM ) and a low binding capacity (2.0 pmol somatostatin/mg protein) and a second class with a low affinity ( K d = 222 nM ) and a high binding capacity (114.3 pmol somatostatin/mg protein). Vasoactive intestinal peptide, neurotensin, substance P, Leu-enkephalin and vasopressin had practically no effect on somatostatin binding. The properties of these binding sites strongly support the concept that somatostatin could behave as a regulatory peptide on the rabbit kidney.

Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa.

Specific binding sites for somatostatin have been found in the cytosolic fractions of both parietal and non-parietal cells from rabbit gastric fundic mucosa. The stoichiometric data suggested the presence of two classes of binding sites in both types of cells. The number of low-affinity binding sites was significantly higher in parietal cells than in non-parietal cells. The reverse was true for the high-affinity binding sites. However, the affinity of each class of binding sites was similar in the cytosolic fractions of both parietal and non-parietal cells. It thus appears that low-affinity somatostatin binding sites are mainly located in the parietal cells whereas the high-affinity sites occur principally in the non-parietal cells.

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa.

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.

Somatostatin binding sites in cytosolic fraction of rabbit intestinal mucosa: Distribution throughout the intestinal tract

1. 1. Specific binding sites for somatostatin have been identified in cytosolic fraction of both small and large intestinal mucosa. 2. 2. The stoichiometric data suggested the presence of two classes of binding sites in each part of the intestine. 3. 3. The binding capacity varied depending on the segment considered (rectum > duodenum = jejunum > ileum, caecum and colon). 4. 4. However, the affinities of the binding sites were similar throughout the whole intestinal mucosa, with the exception of rectum which showed higher K d values. 5. 5. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as vasoactive intestinal peptide, neurotensin, substance P and Leu-enkephalin did not show any effect upon somatostatin binding.

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa.

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.