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Extracts of rat pancreas contain significant amounts of an [3H]estradiol-binding protein. The amount of steroid-binding activity that could be measured varied considerably depending on the tonicity of the homogenizing medium. High speed supernatants of homogenates initially prepared in isotonic buffer contained about 10% of the binding activity as homogenates prepared in hypotonic buffer. Extraction with hypotonic buffer of pellets obtained by the isotonic procedure yielded most of the remaining [3H]estradiol-binding activity. In an attempt to avoid errors resulting from incomplete homogenization and to detect possible changes in intracellular distribution of [3H]estradiol-binding activity, pancreata were initially homogenized in isotonic buffer and centrifuged at high speed (100,000 X g; 1 hr). The pellet was then extracted with hypotonic buffer and centrifuged again at high speed, and both supernatants were analyzed for [3H]estradiol-binding and amylase activities. Two or 14 days after treatment of male rats with streptozotocin, no apparent decline or redistribution of [3H]estradiol-binding activity to the cytosol was noted despite extensive alteration of beta-islet cells, as determined by electron microscopic examination of sections of these pancreata and significant loss of insulin, as measured by RIA. Amylase activity was unaffected 2 days after streptozotocin treatment, but was depressed to about 1% of control levels at 14 days. Administration of insulin to the latter group of animals resulted in return of amylase to normal levels and a modest increase (approximately 50%) in [3H]estradiol-binding activity. Since amylase levels remained unchanged 2 days after streptozotocin treatment, during which time beta-islet cells were irreversibly altered, and amylase activity was restored to normal levels by insulin treatment after its depletion in chronically treated animals, it follows that neither amylase nor the [3H]estradiol-binding protein could have been associated with beta-islet cells. This was consistent with the observation that M cells (a tumor line of beta-cells only) and 14B cells (a cloned variant of this insulinoma) had neither detectable amounts of amylase nor [3H]estradiol-binding activity. To determine whether estrogen-binding activity was associated with any other type of islet cell, islets of Langerhans were isolated by the sedimentation procedure of Lacy and Kostianovsky. In this procedure, several washing steps are employed to separate the suspended acinar cells from the denser islets that sediment rapidly. During this isolation procedure, the cells from each wash were analyzed for protein, [3H]estradiol-binding protein, and amylase a

There is present in rat pancreas a protein that requires an accessory factor in order to bind [3H]estradiol. To identify this accessory factor 874g of dog pancreas were acid extracted, and following selective filtration and dialysis, the low molecular weight constituents (less than 10,000) were concentrated by lyophilization. Samples of this lyophilizate were fractionated by high performance liquid chromatography (HPLC) and eluate fractions analyzed for their capacity to enhance binding of [3H]estradiol to a protein fraction from rat pancreas that had been purified relatively free of endogenous accessory factor. Such enhancement of [3H]estradiol-binding activity eluted predominantly in one peak that coincided with the elution profile of pure somatostatin (SRIF14). Analysis of eluate fractions for somatostatin-like immunoreactive material (SLIM) indicated coincidence of SLIM with the factor that enhanced binding of [3H]estradiol. It appears likely that accessory factor in pancreas is primarily somatostatin (SRIF14). Following incubation of [125I]SRIF14 and [3H]estradiol with a partially purified binding-protein fraction from rat pancreas, a complex containing labeled [125I] and [3H] was separated by Sephadex G-200 column chromatography. In the presence of 25 microM SRIF14, which activates [3H]estradiol-binding maximally in the presence of 10 nM steroid, a protein peak containing both radiolabeled ligands eluted in the void volume indicating an apparent molecular size in excess of 200,000 Daltons. At a concentration of 1 microM SRIF14, a complex eluted at a position corresponding to an apparent Mr of 120,000. Evidently, the steroid and polypeptide mutually enhance binding to this pancreatic protein, and depending on their concentrations form structures of widely varying sizes.

Studies have previously demonstrated low somatostatin levels in autopsy cortical tissue from Alzheimer's disease (AD) patients and low somatostatin levels in CSF obtained from subjects with dementia. We evaluated levels of this peptide in 21 non-depressed subjects, 10 with AD, 2 with Parkinson's disease (PD), and 9 with other neurological conditions. The AD patients had significantly lower mean CSF somatostatin than the 'other' neurological patients (14.6 +/- 1.5 S.E.M. versus 26.7 +/- 3.2 pg/ml, p less than 0.005). A demented PD subject had a level in the range of the AD group, while the non-demented PD patient had a value above this range. Thus, all 11 patients with AD or PD dementia, analogous disorders, had levels below 21.8 mg/ml, while 7 of the 10 remaining patients had values above 21.8 pg/ml. Age did not explain this finding

The cytosol fraction of rat pancrease can bind [3H] estradiol specifically and extensively. In contrast to the rat uterus, the binding protein in pancreas requires an accessory factor as a coligand in the steroid-binding reaction. Removal of this accessory factor by passage of the cytosol through CM Affi-Gel blue columns renders eluate fractions virtually incompetent with respect to binding of [3H]estradiol (10 nM). Certain synthetic oligopeptides such as N-benzoyl-L-argininyl-p-nitroanilide, as well as an endogenous accessory factor, can reactivate binding of [3H]estradiol. Thus, localization of the protein that binds [3H]estradiol following chromatography with CM Affi-Gel blue columns can be determined readily by assaying eluate fractions in the absence and presence of either accessory factor or N-benzoyl-L-argininyl-p-nitroanilide. Addition of somatostatin (tetradecapeptide referred to as SRIF14; somatotropin release inhibiting factor) to the activatable, but incompetent, eluate fractions, also enhanced binding of [3H]estradiol. The effect of SRIF14 was biphasic. The threshold concentration required for activation of [3H]estradiol binding was about 1 microM, and maximal stimulation occurred at 25 microM. At higher concentrations of SRIF14, binding declined and reached basal levels at about 75 microM. The concentrations of somatostatin required for activation of binding of [3H]estradiol in vivo may be lower than those indicated above since 1) preparations containing [3H]estradiol-binding protein also contained an SRIF14 peptidase. Following incubation of [125I-Tyr1]SRIF14 with these preparations there was loss of binding of radiolabeled peptide with SRIF14 antiserum. 2) The biphasic nature of SRIF14 activation may reflect feedback inhibition of [3H]estradiol binding by a degradation product of SRIF14. Since SRIF14 has been identified in the delta- (or D-) islet cells of the pancreas, and in concentrations that may be in the microM range, the possibility is raised that these cells serve a paracrine function with respect to acinar cell secretion

We studied the release of immunoreactive somatostatin (IR-SRIF) from hypothalamic cells that were obtained from rats and dispersed with the aid of collagenase. Twenty-four hours after dispersion, cells were placed in a column supported by a matrix of preswollen Biogel P2 and perifused. Fractions were collected on ice and subsequently assayed for SRIF. SRIF release was stimulated markedly by potassium depolarization (KCl, 56 mM), by the Na+-K+-ATPase inhibitor ouabain (10(-4) M), and by dopamine at concentrations as low as 10(-11) M. The stimulatory effects of membrane depolarization were calcium dependent and were not observed in the absence of exogenous calcium in the perifusion medium or in the presence of EDTA (0.05 M). Metoclopramide, the dopamine antagonist, abolished the stimulatory effect of dopamine. In conclusion, release of IR-SRIF by dispersed rat hypothalamic cells can be studied in a simple perifusion apparatus. Release is stimulated by membrane depolarization in a calcium-dependent manner and by dopamine at physiological concentrations

We have developed a new short term in vitro system to examine hypothalamic somatostatin (SRIF) release. Hypothalamic cells were obtained from normal rats after trypsin or collagenase aided dispersion and released immuno-reactive (IR) SRIF which eluted in 3 molecular weight (MW) forms on gel chromatography. The smallest MW form, which constituted the major peak, co-eluted with synthetic cyclic 1-14 SRIF on gel and reverse phase high pressure liquid chromatography (HPLC). After 24 h in culture in medium containing heat inactivated fetal calf serum, cell viability was demonstrated by two techniques, (1) vital staining with trypan blue, and (2) incorporation of 32Pi into phospholipids. SRIF release was also studied at this time which was optimal in terms of responsivity of the cells to depolarizing stimuli. SRIF release increased in a time dependent manner, over 3 h. Membrane depolarization, induced either by potassium chloride 56 mM or ouabain (the Na+, K+-ATPase inhibitor) 10(-6) M or greater, markedly stimulated SRIF release. Incubation at 4 degrees C, or in the presence of EDTA 0.05 M or verapamil, the calcium channel blocker, 50 microM abolished these stimulatory effects. Glucose deprivation was induced by the addition of 2-deoxy-D-glucose (2-DG) to the experimental medium. 2-DG, at concentrations of up to 200 mg%, had no significant effect on SRIF release during incubation periods of up to 1 h

We have examined the release of radioimmunoassayable luteinizing hormone-releasing hormone (LH-RH) from fragments of rat medial basal hypothalamus. These fragments were cultured overnight in medium containing serum and then preincubated in groups of three for 10 min in medium resembling cerebrospinal fluid in its electrolyte constituents and containing bacitracin. This was followed by 30-min incubation periods during which some of the hypothalami were exposed to test substances. Potassium depolarization, effected by the addition of 56 mM potassium chloride to the incubation medium, caused a marked stimulation in LH-RH release, but only in the presence of calcium. Acetylcholine at 10 nM and the parasympathomimetic anticholinesterase agent neostigmine at 1 microM markedly stimulated LH-RH release. Hexamethonium, a nicotinic antagonist, at 1 microM abolished the acetylcholine-induced increment in LH-RH release. Melatonin, a pineal indolamine, caused significant stimulation of LH-RH release at a concentration as low as 10 nM. Bacitracin (21 microM) was employed in all these experiments. It had no effect on LH-RH release but did prevent the degradation of LH-RH in this system. We conclude that acetylcholine and melatonin are capable of inducing LH-RH release from the rat medial basal hypothalamus. These actions may account for some of the progonadotropic properties previously ascribed to these agents