Ontogeny of immunoreactive CCK,VIP and secretin in rat brain and gut

Immunoreactive cholecystokinin (iCCK), vasoactive intestinal peptide (iVIP) and secretin (iSEC) were determined in the brain and various gut regions in the developing rat between 3 and 28 days after birth and in the adult. From the different patterns observed with these three peptides, it is concluded that in rat neural tissues, peptide concentrations (iCCK in brain, iVIP in brain and gut) increase continuously until about 4 weeks. Concentrations in mucosal tissues (iSEC in gut) are equal to or higher than adult values 3 days after birth. Gut iCCK (found both in neuronal and mucosal tissues) peaks at about 2 weeks, presumably due to concentrations increasing in the former and decreasing in the latter tissues.     

Multiple molecular forms of rat brain enkephalinase

Rat brain enkephalinase has been partially purified by ion exchange chromatography, chromatofocusing, and affinity chromatography on immobilized lectins. Ion exchange chromatography resolved two principle forms of enkephalinase designated A1 and A2. Both enkephalinase A1 and A2 are bound to immobilized lentil lectin while chromatography on immobilized wheat germ lectin resolved each of the principle forms into two subforms, A1, 1, A1, 2, A2, 1, and A2, 2. All four enkephalinase forms have similar, if not identical kinetic properties. The possible implications of multiple molecular forms of enkephalinases are discussed.     

Preferential inhibition of acetylcholinesterase molecular forms in rat brain

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G₄), and monomeric (G₁) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G₁ than for the G₄ form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G₁ form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.Special issue dedicated to Dr. Morris H. Aprison.     

Ontogeny of immunoreactive CCK and VIP in pig brain and gut

The concentrations and hormonal forms of CCK and VIP have been determined in extracts of the brain and duodenum of the developing and adult pig. In methanol extracts of the brain cortex, the single hormone form, CCK8, increased from 130 +/- 20 (Mean +/- SEM) pmol/g at birth to an adult level of 300 +/- 50 pmol/g. In acid extracts of brain, the predominant immunoreactive form had N-terminal immunoreactivity and increased from 240 +/- 20 pmol/g at birth to an adult level 490 +/- 30 pmol/g; the C-terminal immunoreactivity was about 10-fold lower. The concentrations and hormonal forms of immunoreactive CCK in duodenal extracts did not appear to be age-related. C-terminal immunoreactivity in methanol extracts averaged 140 +/- 20 pmol/g and in acid extracts 240 +/- 60 pmol/g. The concentration of N-terminal immunoreactivity in acid extracts averaged 490 +/- 70 pmol/g. The VIP concentrations in acid extracts of the brain cortex was 13.5 +/- 2 pmol/g at birth and rose gradually to 30 +/- 9 pmol/g in the adult; in duodenal extracts it was 240 +/- 18 pmol/g at birth and 195 +/- 38 pmol/g in the adult. These results are in marked contrast with the ontogeny of these hormones in the rat in which brain concentrations of CCK and VIP in the neonate are less than 10% of adult levels and in which there are age-related changes in the content of these hormones in the duodenum as well.     

Concentration and molecular forms of brain natriuretic peptide in rat plasma and spinal cord

To characterize the biological functions of rat brain (B-type) natriuretic peptide (BNP), which has been shown to be present mainly in the heart and only faintly in the spinal cord, the concentration and molecular forms of BNP in plasma and spinal cord were determined. The concentration of immunoreactive (ir-) BNP was 2.00 fmol/ml in normal rat and 13.29 fmol/ml in morphine-treated rat, being respectively about 1/20 and 1/80 those of ir-atrial (A-type) natriuretic peptide (ANP). In morphine-treated rats, ir-BNP was shown to circulate mainly as BNP-45, which is identical to a major storage form found in cardiac atrium. In the spinal cord, BNP was also shown to be present as BNP-45, but its concentration was only 0.057 pmol/g, being about 1/60 that of spinal cord ANP. These results confirm that BNP mainly functions as a circulating hormone in the molecular form of BNP-45. Morphine stimulates secretion of ANP and BNP but by different ratios, suggesting different regulation systems for storage and secretion of ANP and BNP.     

Ontogeny of endothelin and its receptors in rat brain.

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Ontogeny and distribution of cysteine sulfinate transaminase in the rat brain

Cysteine sulfinate transaminase catalyzes the conversion of cysteine sulfinate and -ketoglutarate to -sulfonyl pyruvate and glutamate and is present in high concentration in neuronal tissue. During development, levels of cysteine sulfinate transaminase in whole rat brain homogenate increase twofold between days 10 and 21. At maturity, high levels of the enzyme are present in the synaptosomal fraction. The highest level of enzyme activity was in the hypothalamus, approximately three- to fourfold greater than any other region examined. These data suggest that cysteine sulfinate transaminase may have an important role in the metabolism of neurotransmitters and their precursors.     

Similar high molecular weight forms of growth hormone-releasing hormone are found in rat brain and testis

We have utilized a new radioimmunoassay for rat growth hormone-releasing hormone (GHRH) to investigate the presence of GHRH in different organ systems of adult rat, and specifically the rat central nervous system (CNS). The highest concentration of GHRH was found, as expected, in the hypothalamus, but significant amounts were also located in the brain cortex, predominantly the frontal cortex, as well as in the testis. Smaller amounts were identified in the cerebellum and brain stem. Sephadex and reversed phase high performance liquid chromatography demonstrated that while hypothalamic GHRH exclusively eluted at the position of rat GHRH (1-43), in testis and brain the major form was predominantly (testis) or wholly (brain) of a higher molecular weight. While this molecular species has yet to be further characterized, the data suggest the similar GHRH-like species exist in the CNS as well as the testis.     

Ontogeny and distribution of specific succinic semialdehyde reductase apoenzyme in the rat brain

The ontogeny and distribution in rat brain of specific succinic semialdehyde reductase is described. This enzyme is probably responsible for the synthesis of -hydroxybutyrate in brain. The highest activities and levels of apoenzyme are found in cerebellum, olfactory bulb, septum and median hypothalamus. During neonatal development, the enzyme activity remains stable at least until 63 days of age. As the levels of other enzymes of the GABA shunt pathway increase during this same period, this result indicates that there is a relative decrease in the reductive pathway of succinic semialdehyde catabolism during development leading to -hydroxybutyrate synthesis, compared to the oxidative pathway leading to succinate.     

Immunochemical differences among molecular forms of acetylcholinesterase in brain and blood

Molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) differ in their solubility properties as well as in the number of their catalytic subunits. We used monoclonal antibodies to investigate the structure of acetylcholinesterase forms in brain, erythrocytes and serum of rats, rabbits and other mammals. Two antibodies were found to bind tetrameric acetylcholinesterase in preference to the monomeric enzyme. These antibodies also displayed lower affinity for certain forms of 'soluble' brain acetylcholinesterase than for the 'membrane-associated' counterparts. Furthermore, one of them was virtually lacking in affinity for the membrane-associated enzyme of erythrocytes. The basis for the antibody specificity was not fully determined. However, the immunochemical results were supported by measurements of enzyme thermolability, which showed that the catalytic activity of 'soluble' acetylcholinesterase was comparatively heat-resistant. These observations point toward structural differences among the solubility classes of acetylcholinesterase.     

Multiple forms of immunoreactive dynorphin in rat pituitary and brain

Multiple forms of immunoreactive dynorphin (I-dynorphin) in rat anterior pituitary (AP), intermediate-posterior pituitary (IP) and medial basal hypothalamus (MBH) were examined. Three I-dynorphin peaks were observed on gel filtration. The first peak (big form) was eluted near the position of beta-LPH, and was predominant in AP. The second peak (middle form) was eluted near the position of ACTH. The third peak (small form) was eluted at the position of dynorphin (1-13), ane was predominant in IP and MBH. The heterogeneity of this small form was examined by ion exchange chromatography and reverse phase high performance liquified chromatography (HPLC). I-dynorphin peaks were observed at the positions of dynorphin(1-17), (1-13), (1-11), (1-10) and other peptides. These results strongly suggest (i) the presence of dynorphin(1-17), (1-13), (1-11) and (1-10) in rat IP, (ii) dynorphin(1-11) and (1-10) as the major components in this small form, (iii) the difference of I-dynorphin processing in AP, IP and MBH.     

Identification of diverse molecular forms of GnRH in reptile brain

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln⁸-GnRH and His⁵,Trp⁷,Tyr⁸-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His⁵,Trp⁷,Tyr⁸-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp⁷,Leu⁸-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln⁸-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.     

In vivo and in vitro effects of diisopropyl fluorophosphate and paraoxon on individual molecular forms of rat brain acetylcholinesterase

Previous study in this laboratory showed that following a sc injection of an organophosphorus compound, diisopropyl fluorophosphate (DFP), into rats the inhibition of 10S molecular forms was considerably more pronounced than that of 4S forms of brain acetylcholinesterase (AChE). This could depend on different accessibility of the two forms or on their different intrinsic sensitivity to the antiChE compound. In the present study the effects of DFP and Paraoxon on 10S and 4S forms were evaluated in vivo, i.e., after systemic administration, and in vitro by adding the organophosphorus compounds to each of the two forms after extraction from brain of untreated rats, solubilization and separation. The in vivo preferential inhibition of 10S forms was confirmed. The 10S/4S ratios for control and DFP-treated rats were 9.05 and 5.01, respectively; these ratios were 8.46 and 3.33 for Paraoxon. On the other hand, in the in vitro experiments there were no significant differences between IC50 values for 10S and 4S forms both in the case of DFP (2.66 and 2.98 uM) and Paraoxon (32.4 and 42.4 nM, respectively). The overall data suggest that the preferential in vivo inhibition of 10S molecular forms with respect to 4S forms depends on their different accessibility probably due to different subcellular localization of the two forms and not on their different intrinsic sensitivity.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Three molecular forms of a rat asialoglycoprotein receptor

It has been reported that a rat asialoglycoprotein receptor is composed of three polypeptide chains with molecular masses of 43, 54, and 64 kilodaltons (43, 54, and 64-Kd forms) and that the first has a different primary structure from the latter two forms. Incorporation of [3H]leucine into these forms showed that no precursor-product relationship is found between the 54-Kd and 64-Kd forms. The half-life of the 43-Kd form (25 h) was shorter than those of the 54-Kd and 64-Kd forms (66 and 70 h, respectively). Glycopeptides of the three forms were prepared from rat livers previously labeled in vivo with [3H]glucosamine. Gel filtration analysis of the glycopeptides before and after endo H treatment revealed that they were all resistant to endo H. Alkali treatment did not change the elution position appreciately. These results indicate that the three molecular forms contained only complex oligosaccharide chains. The receptor was prepared from rat livers previously treated with tunicamycin in vivo and subjected to SDS-PAGE. A distinct band with a molecular mass of 33 Kd was observed. The receptor was also immunoprecipitated from rat hepatocytes in primary culture previously labeled with [35S]methionine and analysed by SDS-PAGE and fluorography. In addition to the major 43-Kd form, a band with a molecular mass of 41 Kd was found and tunicamycin treatment gave rise to a 33-Kd component, which is in good agreement with the receptor purified from tunicamycin treated rats. It is suggested that the 43-Kd form is synthesized as a 33 Kd polypeptide, cotranslationally glycosylated to form the 41 Kd component and then processed to the final 43-Kd form. We also think that the 43-Kd form could bind to asialoorosomucoid-Sepharose 4B without its carbohydrate chains.