High angiotensin-converting enzyme activity in the neurohypophysis of Brattleboro rats.

The activity of angiotensin-converting enzyme is significantly higher in the intermediate and posterior pituitary lobes of Brattleboro rats than in Long-Evans control rats. The high activity level was reversed by vasopressin treatment. Conversely, angiotensin-converting enzyme activity was significantly lower in the anterior pituitary of Brattleboro rats than in Long-Evans rats, and this activity level was not affected by vasopressin. these findings suggest an inverse relation between vasopressin and angiotensin systems in the posterior and intermediate lobes of the pituitary gland.

Adenosine triphosphate and adenosine triphosphatase in hormone-containing granules of posterior pituitary gland.

Neurosecretory granules prepared from bovine posterior pituitary glands by cell fractionation methods contain adenosine triphosphate and adenosine triphosphatase activity. Addition of adenosine triphosphate to suspensions of granules stimulates release of vasopressin. It is suggested that adenosine triphosphate and adenosine triphosphatase participate in the storage and release of vasopressin.

Activation of multifunctional Ca2+/calmodulin-dependent kinase in intact hippocampal slices.

In vitro phosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) converts it to a form that is independent of Ca2+. We demonstrate that significant Ca(2+)-independent CaM kinase activity is present in untreated hippocampal slices. Two manipulations that produce a long-lasting enhancement of neuronal activity in hippocampal slices, elevated extracellular Ca2+ or depolarization with high K+, generate additional Ca(2+)-independent activity. This increase is dependent on extracellular Ca2+ and is correlated with an increased phosphorylation of CaM kinase. In contrast, CaM kinase in posterior pituitary, a brain structure that is not thought to be involved in memory-related processes, is not modulated by depolarization. These results suggest that the Ca(2+)-independent form of CaM kinase may modulate neuronal activity in the hippocampus.

Cytidine-5'-monophospho-N-acetylneuraminic acid galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide sialyltransferase in the neurohypophysis of the rabbit.

1. Cytidine-5'-monophospho-N-acetylneuraminic acid: (galactosyl-N-acetyl-galactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide sialyltransferase (CMP-NAcNeu: monosialoganglioside (GM1) sialyltransferase) activity was demonstrated in the neurohypophysis of the rabbit. 2. Optimum activity occurred at pH 6.5 and required the presence of exogenous galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide (GM1 ganglioside), detergent (Triton X-100), and divalent cation (Mn2+, Mg2+ or Ca2+). 3. The product of the reaction was characterized as N-acetylneuraminyl-galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosylceramide (GD1a) by ascending thin-layer chromatography. 4. Physiological stimulation of vasopressin secretion, by the substitution of 2.2% NaCl for drinking water for 14 days, had no effect on the enzyme activiity or the ganglioside content of the tissue.

Subcellular fractionation of pituitary neurointermediate lobes: revelation of various basic proteases.

Homogenates of rat neurointermediate lobes were purified by centrifugation on a Percoll gradient. Lysates of the Percoll gradient fractions were incubated with a synthetic octapeptide and pentapeptide substrate (N-acetyl Lys-Arg-Tyr-Asn-Leu-Thr-Ser-Val-amide and N-acetyl Lys-Arg-Tyr-Asn-Leu-amide), and enzymatic characteristics were compiled. Early assays on nonamidated forms revealed carboxypeptidase activity, whereas with the amide derivatives no carboxypeptidase activity could be detected. These amide substrates were therefore used in all subsequent incubations. High levels of a Leu/Thr and Lys/Arg cleavage were present in fractions almost throughout the Percoll gradient. Cleavages at Tyr/Asn, Thr/Ser, and Arg/Tyr were localized at different regions of the Percoll gradient. Surprisingly, none of the five enzymatic activities appear to be localized in the secretory granule fractions as defined by the presence of immunoreactive beta-endorphin in the gradient. All of the five proteolytic activities have a basic pH optimum (pH 8-9), and four of them seem to be thiol proteases, as categorized by inhibitor studies. The fifth one, namely the Try/Asn cleavage, is more likely to be due to a metalloendopeptidase, since it is activated by Zn2+ and Co2+.

[3H]spiroperidol identifies a D-2 dopamine receptor inhibiting adenylate cyclase activity in the intermediate lobe of the rat pituitary gland.

[3H]Spiroperidol ([3H]SPIRO) binds with high affinity (Kd = 0.3 nM) to cell-free homogenates of the neurointermediate lobe of the rat pituitary gland. The neurointermediate lobe contains 19.2 fmol of specific binding sites, 86% of which occur in the intermediate lobe (IL). Compounds active upon the D-2 dopamine receptor in the IL compete with [3H]SPIRO for occupancy of the specific binding site. Guanosine 5'-triphosphate decreases the affinity of agonists, but not antagonists, for the specific binding site. For each drug tested, methods derived from competitive enzyme kinetics were used to calculate the apparent affinity constants of the drug for the binding site and for the receptor regulating adenylate cyclase activity. The pharmacological properties of the specific [3H]SPIRO binding site were compared to the pharmacological properties of the D-2 dopamine receptor inhibiting adenylate cyclase activity in the IL. The similarity between the affinities determined from the binding and enzyme assays suggests that some or all of the specific [3H]SPIRO binding sites in the IL are D-2 dopamine receptors inhibiting adenylate cyclase activity.

Extended chain analogs of [arginine8]vasopressin as model prohormones: investigation of precursor-processing enzymes in extracts of the rat hypothalamus and neural lobe.

Analogs of [arginine8]vasopressin (AVP) in which the peptide chain was elongated from the N-terminus by the addition of Ala-Arg-Arg-, Ala-Ala-Phe-, Pro-Arg-Val-, Pro-Ala-Arg-Arg, and Pro-Ala-Ala-Phe-, and from the C-terminus by the addition of -Ala-Met-Ala-NH2 and -Gly-Arg-Arg-Ala-NH2 were synthesized by the solid phase method and purified by Sephadex G-15 chromatography. At the final step of the synthesis, the extent of formation of the intramolecular disulfide bond was found to be sequence dependent. These peptides were incubated with extracts of the rat hypothalamus (supraoptic region) and neural lobe and with isolated neurosecretory granules from the neural lobe, and the release of vasopressin was measured by the rat pressor assay. All peptides resisted conversion to the hormone in the presence of tissue extracts, except (Ala-Ala-Phe)-AVP which was converted to AVP in the presence of all three tissue extracts at pH 4.7 but not at pH 8.0. When these peptides were treated with trypsin, chymotrypsin, or leucine aminopeptidase at pH 8.0, only the action of chymotrypsin on [Ala-Ala-Phe]AVP resulted in AVP formation. Evidence obtained using lysosomal enzyme markers suggested that the converting enzyme activity in neurosecretory granule preparations was not of lysosomal origin.

Tyrosine hydroxylase in the stalk-median eminence and posterior pituitary is inactivated only during the plateau phase of the preovulatory prolactin surge.

This study examined changes in the activity of tyrosine hydroxylase (TH) in the stalk-median eminence (SME) and posterior pituitary (PP) during the preovulatory PRL surge. Immature female rats were injected with PMSG on day 28. Blood PRL levels were low on the morning of day 30, rose to a peak from 1400-1600 h, remained at a lower plateau from 1800-2400 h, and declined to basal levels on the morning of day 31. SME, PP, and striatum were removed from PMSG-treated rats at selected times during the periovulatory period and from age-matched control rats. TH activity was determined in tissue homogenates by a coupled hydroxylation-decarboxylation assay. Apparent Km and maximum velocity values with respect to 6-methyl tetrahydropterine were estimated from substrate saturation curves. The kinetic parameters for TH in either the SME or the PP of control rats were similar at 1100 and 1800 h on day 30. However, the apparent Km in both tissues was significantly lower than that in the striatum. The affinity of TH in the SME and PP was unchanged before and during the peak phase of the PRL surge, reduced significantly during the late plateau, and returned to presurge levels in the morning of day 31. TH activity in the striatum was similar at all times examined. To determine the state of activation of the enzyme, tissue homogenates were preincubated with cAMP, ATP, and magnesium. TH activity in the SME during the peak phase was unchanged by cAMP, and that in the PP was modestly increased. The relatively inactive enzyme in both tissues during the plateau phase was markedly activated by a cAMP-dependent mechanism. The low affinity of striatal TH was greatly increased by cAMP at both times. These data suggest that TH in the SME and PP exists in an activated state most of the time and is transiently inactivated during the plateau phase of the PRL surge. In contrast, TH in the striatum is relatively inactive in the basal state and is not affected by hormonal changes induced by PMSG. We conclude that the peak PRL surge occurs in spite of active dopamine (DA) neurons, suggesting that it is generated by a nondopaminergic mechanism. Decreased TH activity in DA neurons in the SME and PP may prolong the PRL surge during the plateau phase, whereas increased DA activity coincides with the termination of the surge.

Role of peptide substrate structure in the selective processing of peptide prohormones at basic amino acid pairs by endoproteases.

Three putative processing enzymes, each with defined action in a prohormone system, a 'pro-ocytocin-neurophysin convertase' from bovine neurohypophysis secretory granules, a 'Leu-enkephalin Arg6 generating enzyme' from human CSF and the endoprotease from the 'S-28 convertase' complex of rat brain cortex, were tested for their ability to hydrolyze peptides deriving from pro-ocytocin, pro-enkephalin B and pro-somatostatin, respectively at pairs of basic amino acids. The observations suggest that structural parameters specified by the peptide region around the dibasic moieties govern recognition by the enzyme and define which peptide bond is hydrolyzed.

An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin60-65.

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.

Differentiation of tyrosine hydroxylase-synthesizing and/or aromatic L-amino acid decarboxylase-synthesizing neurons in the rat mediobasal hypothalamus: quantitative double-immunofluorescence study.

In this double-immunofluorescence study, we first quantified the neurons of the arcuate nucleus as immunoreactive (+) for tyrosine hydroxylase (TH) and/or aromatic L-amino acid decarboxylase (AADC) in rats at embryonic day 21 (E21), at postnatal day 9 (P9), and in adulthood by using conventional fluorescent or confocal microscopy. On E21, monoenzymatic (TH(+)AADC immunonegative (-) and TH(-)AADC(+)) neurons and bienzymatic (TH(+)AADC(+)) neurons accounted for 99% and 1%, respectively, of the whole neuron population expressing enzymes of dopamine synthesis. Further development was characterized by the dramatic increase in TH(+)AADC(-) dorsomedial and TH(+)AADC(+) dorsomedial populations from E21 to P9 as well as by the increase in the TH(+)AADC(+) dorsomedial population (in females) and a drop in the TH(+)AADC(-) ventrolateral and TH(+)AADC(-) dorsomedial (in males) populations from P9 to adulthood. In contrast to TH(+)AADC(-) (in males) and TH(+)AADC(+) neurons, the TH(-)AADC(+) neurons did not change in number from E21 to adulthood. Thus, in rat fetuses, the neurons synthesizing TH and/or AADC were mainly monoenzymatic, whereas during postnatal life the fraction of bienzymatic neurons increased by up to 60%.

Rat pituitary N alpha-acetyltransferase.

The rat pituitary contains an enzyme which will acetylate certain ACTH fragments using acetyl coenzyme A. This acetyltransferase activity was found in all three lobes of the rat pituitary as well as in all other tissues examined. The rat pituitary enzyme appears to be largely particulate in nature. The enzyme sedimenting at 27,000 and 100,000 g had specific activities 4-10 times greater than the soluble fraction. The acetyltransferase activity was dependent on substrate concentration (ACTH) and pH, was linear with time, and was inactivated at 55 degrees. The enzyme would acetylate ACTH(1-24), (1-10), and ACTH(4-10), but would not use ACTH(2-10), (3-10), or ACTH(1-8) as substrates. The apparent Km values for the substrates were as follows: AcCoA, 2.2 microM, ACTH(1-24), 4.2 microM; ACTH(1-10), 96 microM; and ACTH(4-10), 37 microM.

Acetylcholinesterase in the rat neurohypophysis is decreased after dehydration and released by stimulation of the pituitary stalk.

Specific acetylcholinesterase and non-specific cholinesterases are present in all three lobes of the rat pituitary gland. This paper describes two new observations on hypophyseal acetylcholinesterase. Firstly, a prolonged increase of neurohormone secretion evoked by dehydration and sodium loading was accompanied by a decrease in the acetylcholinesterase activity localized to the neural lobe, where acetylcholinesterase has previously been demonstrated in fine nerve fibres. Secondly, electrical stimulation of the pituitary stalk in vitro elicited the release of acetylcholinesterase and non-specific cholinesterases from the combined neural and intermediate lobe indicating that the enzyme can be released from nerve endings in the hypophysis by action potentials. The observed loss of enzyme activity during dehydration may be the consequence of a prolonged activation of cholinergic nerves in the gland, leading to an increased release of acetylcholinesterase, which is not immediately replaced by fresh enzyme. The decrease in acetylcholinesterase in the neural lobe during dehydration may also be connected with its peptidase function and thus with the previously observed loss of substance P from the neural lobe during dehydration [Holzbauer et al. (1984) Neurosci. Lett. 47, 23-28].

Aminopeptidases in the circumventricular organs of the mouse brain: a histochemical study.

The localization of four membrane-bound aminopeptidases--aminopeptidase A, aminopeptidase M, dipeptidylpeptidase IV, and gamma-glutamyl transpeptidase--known as characteristic enzymes of the blood-brain barrier was studied in the microvasculature of some circumventricular organs of the mouse brain (subfornical organ, area postrema, choroid plexus, and neurohypophysis). Enzyme activities were demonstrated histochemically in chloroform-acetone-pretreated cryostat sections applying an azo-coupling method. Reactions were evaluated using light microscopy and end-point microdensitometry. The results revealed differences in microvascular enzyme pattern between circumventricular organs and regions having a blood-brain barrier. Moreover, the cytochemical picture of the circumventricular organs themselves was not uniform. Dipeptidylpeptidase IV reaction showed a strongly reduced activity in the microvessels of all studied circumventricular organs. On the other hand, aminopeptidase M seemed to be present in both the leaky and the tight capillaries. Only a low activity of aminopeptidase A was found in parts of the choroid endothelium and the subfornical organ microvasculature. gamma-Glutamyl transpeptidase could neither be detected in the capillary part of the choroid plexus nor in the neurohypophysis. We are led to conclude that at least dipeptidylpeptidase IV might be involved in special mechanisms of the blood-brain barrier.

Effects of oestrogen upon nitric oxide synthase NADPH-diaphorase activity in the hypothalamo-neurohypophysial system of the rat.

An understanding of the interaction between oestrogen and the nitric oxide synthase/nitric oxide system is important for determining the roles of nitric oxide in central nervous control of osmotic homeostasis and certain aspects of reproduction. The effects of oestrogen on nitric oxide synthase and nitric oxide synthase activity were investigated in the magnocellular neurosecretory system. Ovariectomized female rats were injected subcutaneously with 17beta-estradiol benzoate either 10 microg daily for four days (short-term low-dose) or 200 microg daily for 21 days (long-term high-dose). In the neurohypophysis the density of NADPH-diaphorase staining--a marker for nitric oxide synthase activity--was increased after both short-term low-dose and long-term high-dose estradiol treatment, but no difference in nitric oxide synthase immunoreactivity was observed after either treatment. In the magnocellular supraoptic and paraventricular nuclei, short-term low-dose oestrogen treatment did not induce any detectable changes in nitric oxide synthase gene expression, the proportion of nitric oxide synthase-immunoreactive neurons, or the proportion of NADPH-diaphorase-positive neurons. Long-term high-dose oestrogen treatment also had no effect on nitric oxide synthase gene expression or immunoreactivity, but caused a reduction of the proportion of NADPH-diaphorase-positive neurons in the supraoptic nucleus and a reduction in the intensity of this histochemical staining. Qualitatively similar changes were observed in the magnocellular part of the paraventricular nucleus. The results provide, for the first time, evidence of a complex interaction between oestrogen and nitric oxide synthase in the neuroendocrine system in which nitric oxide synthase activity is regulated differently in the magnocellular cell bodies and axonal terminals and in which the activity of the enzyme rather than its expression is controlled.

Cytochemical localization of ecto-ATPases in rat neurohypophysis.

We studied the distribution of Ca(2+)- or Mg(2+)-dependent ATPase activity in rat neurohypophysis using the lead cytochemical method of Ando et al. In electron microscopy, precipitates were found lining the outer surface of the plasma membrane surrounding nerve endings and pituicytes. These precipitates were believed to represent the activity of ecto-ATPases (as opposed to Ca pump ATPases) for the following reasons: there was equal activation by Ca2+ in the absence of Mg2+ or Mg2+ in the absence of Ca2+; the effects of the two ions were not additive; there was activation by ATP or GTP; and there was resistance to glutaraldehyde fixation, to high (10 mM) Ca2+ concentrations, and to various inhibitors such as NEM, vanadate, oligomycin, quercetin, p-chloromercuribenzoate, ouabain, and levamisole. Cytosolic activity observed in certain nerve endings in the same conditions of incubation but more sensitive to NEM is also described and discussed.

Immunohistochemical localization and comparison of carboxypeptidases D, E, and Z, alpha-MSH, ACTH, and MIB-1 between human anterior and corticotroph cell "basophil invasion" of the posterior pituitary.

Basophil invasion, i.e., invasion of basophilic corticotrophs from the residual intermediate lobe into the posterior lobe of the human pituitary gland, is believed to be a physiological phenomenon. This study evaluated the distribution of CPE, CPD, CPZ, alpha-MSH, ACTH, and Ki-67 immunoreactivity between human anterior pituitary and basophil invasion of the neurohypophysis. Mild to moderate immunoreactivities for CPE and CPZ were distributed relatively uniformly in the majority of the anterior pituitary cells and basophil invasion. In contrast, only corticotrophs exhibited intense CPD immunoreactivity. Basophil invasion showed similar immunoreactivities for alpha-MSH, ACTH, CPE, and CPZ as corticotrophs in the anterior pituitary, except for CPD, which was detected much less frequently. In the posterior lobe, CPE, CPD, and CPZ were present within the Herring bodies. Although no MIB-1 immunoreactivity was identified in anterior pituitary cells, limited MIB-1 labeling was detected in basophil invasion in five of ten cases. Highly selective expression of CPD in corticotrophs suggests that CPD plays a particularly important role in prohormone (POMC) processing in corticotrophs, with minimal or no significant roles in non-corticotrophs. Evidence that corticotrophs in basophil invasion are undergoing proliferation and are also phenotypically different from their counterpart in the anterior pituitary has further raised the possibility of some neoplastic potential.

An uneven distribution of choline acetyltransferase activity in the pituiatry neurointermediate lobe of the rat.

The neurointermediate lobe of the pituitary gland of male rats was assayed for choline acetyltransferase (CAT) activity. Precise microsample punches were obtained from neurohypophysial tissue, pure pars intermedia tissue and from the junction area between them. The level of CAT activity (pmol/h per microgram protein) in the neurohypophysis, pars intermedia and junction area were 0.390 +/- 0.038 (S.E.M.), 0.228 +/- 0.042 and 1.824 +/- 0.268 respectively. These values show an uneven distribution of CAT in the neurointermediate lobe. The hypothesis of a cholinergic system located in the junction area has been advanced.

Dissociation of nitric oxide synthase immunoreactivity and NADPH-diaphorase enzyme activity in rat pituitary.

The relationship between nitric oxide synthase (NOS) immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry was investigated in the anterior and posterior pituitary of ovariectomized rats. NADPH-d activity was present throughout the posterior pituitary but could not be detected in the anterior pituitary. By contrast, an antibody raised against whole recombinant rat neuronal NOS revealed strongly NOS-immunoreactive gonadotrophs and folliculo-stellate cells scattered throughout the anterior pituitary in addition to immunoreactive fibres in the posterior pituitary. The study demonstrates that NADPH-d histochemistry is not always coincident with NOS and provides evidence for an as yet uncharacterized subtype of NOS in the rat anterior pituitary.

Expression of 3 beta-hydroxysteroid dehydrogenase/isomerase in the female rat pituitary.

3 beta-Hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) catalyses an essential step in the biosynthesis of steroid hormones and is widely distributed in peripheral steroid target organs. The present report describes for first time the expression of this enzyme in the pituitary of female rats. Immunohistochemistry at the light microscopic level was performed on pro-oestrous and ovariectomized rat pituitaries. Immunoreactive cells were scattered and randomly distributed throughout the anterior lobe, whereas cells located in the posterior lobe and pars intermedia were immunonegative. Differences were observed in cell morphology and in the number of 3 beta-HSD-immunopositive cells between ovariectomized and pro-oestrous female rat pituitaries, suggesting that steroidogenic activity is affected by ovarian endocrine function. Apart from adenohypophyseal immunoreactive cells, 3 beta-HSD immunopositivity was also noted in endothelial cells of almost all pituitary capillaries located in the anterior and posterior lobes.