Comparative studies on the release of vasopressin and 7B2 from isolated hypothalami

A new protein, 7B2, has been found in the anterior, intermediate and posterior pituitary and the hypothalamus. It has been previously suggested that 7B2 is synthesized in the hypothalamus and is transported to the posterior lobe in a similar way to vasopressin (AVP). We examined the in vitro release of AVP and 7B2, using rat hypothalami in a perifused column system. Membrane depolarization with KCl (56 mM) caused a marked stimulation of AVP release (from 116% to 263% above basal values). Release of 7B2 was also stimulated by potassium from 106% to 212% above basal values. Potassium pulses in calcium-free medium failed to release AVP or 7B2. Osmotic and cholinergic stimulation increased AVP secretion by 205% and 282% above basal values, respectively, but had no significant effect on 7B2 secretion. Chromatographic profiles of perifusion media revealed one immunoreactive 7B2 peak eluting at a coefficient of 0.34 corresponding to that of rat hypothalamic 7B2. Similarly AVP coeluted with AVP standard. Thus AVP and 7B2 are differentially released by cholinergic and osmotic stimuli in vitro.     

Involvement of Accessory Neurosecretory Nuclei of Hypothalamus in the Formation of Hypothalamohypohysial System during Prenatal and Postnatal Development in Rats

We studied the development of direct axonal connections of the accessory neurosecretory hypothalamic nuclei with the posterior pituitary lobe on the fixed rat brain from day 15 of embryogenesis until day 10 of postnatal development using the retrograde diffusion method of the lipophilic fluorescent carbocyanine dye 1,1"-dioctadecyl-3,3,3",3"-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The marker was applied onto the posterior pituitary lobe and, after incubation in a fixative, fluorescing bodies of nerve cells were visualized in the hypothalamus. Neuronal axons of the retrochiasmatic nucleus were the first of the accessory nuclei to ingrow in the posterior pituitary lobe (on days 16–17 of embryogenesis). Neurons of the circular and dorsolateral nuclei and the nuclei of the median bundle of the forebrain sent their axons to the posterior pituitary lobe starting from the first days of postnatal development. No direct connections of the anterior commissure and perifornical accessory nuclei with the posterior pituitary lobe were found in perinatal development. These facts are discussed in the light of concepts about the different functional role of accessory peptidergic hypothalamic nuclei in rats.     

Vasopressin release from posterior pituitary lobe incubated in situ after preganglionic stimulation of the rat superior cervical ganglion.

In urethane-chloralose anaesthesia the pituitary gland was exposed by transpharyngeal approach in rats. The anterior lobe was removed and the posterior lobe was incubated in situ, that is in conditions of anatomical integrity of the hypothalamus with the posterior pituitary lobe. The 15-min samples of the medium incubating the posterior pituitary lobe in situ were collected. Vasopressin (AVP) content in the incubation medium was determined by radioimmunoassay. The stimulation of preganglionic fibers of the superior cervical ganglion (SCG) with alternate short (5 s) bursts of electric pulses with short (5 s) breaks did not change AVP release. However, stimulation of preganglionic fibres with alternate long (30 s) bursts of electric pulses with long (30 s) breaks evoked an increase in AVP release after some latency. Probably, at the hypothalamic or posterior pituitary level temporal summation should occur affecting vasopressinergic neurons or their endings and evoking AVP release.     

Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization

CRF receptors were characterized using radioligand binding and chemical affinity cross-linking techniques and localized using autoradiographic techniques in porcine, bovine and rat pituitaries. The binding of 125I-[Tyr0]-ovine CRF (125I-oCRF) to porcine anterior and neurointermediate lobe membranes was saturable and of high affinity with comparable KD values (200-600 pM) and receptor densities (100-200 fmoles/mg protein). The pharmacological rank order of potencies for various analogs and fragments of CRF in inhibiting 125I-oCRF binding in neurointermediate lobe was characteristic of the well-established CRF receptor in anterior pituitary. Furthermore, the binding of 125I-oCRF to both anterior and neurointermediate lobes of the pituitary was guanine nucleotide-sensitive. Affinity cross-linking studies revealed that the molecular weight of the CRF binding protein in rat intermediate lobe was identical to that in rat anterior lobe (Mr = 75,000). While the CRF binding protein in the anterior lobes of porcine and bovine pituitaries had identical molecular weights to CRF receptors in rat pituitary (Mr = 75,000), the molecular weight of the CRF binding protein in porcine and bovine intermediate lobe was slightly higher (Mr = 78,000). Pituitary autoradiograms from the three species showed specific binding sites for 125I-oCRF in anterior and intermediate lobes, with none being apparent in the posterior pituitary. The identification of CRF receptors in the intermediate lobe with comparable characteristics to those previously identified in the anterior pituitary substantiate further the physiological role of CRF in regulating intermediate lobe hormone secretion.     

Effect of intranasal arginine vasopressin on human headache

Arginine vasopressin (AVP), a nonapeptide hormone of posterior pituitary, reaches the central nervous system from systemic blood circulation with a difficulty because of the blood–brain barrier (BBB). The interest has been expressed in the use of the nasal route for delivery of AVP to the brain directly, exploiting the olfactory pathway. Our previous study has demonstrated that AVP in the brain rather than the spinal cord and blood circulation plays an important role in rat pain modulation. For understanding the role of AVP on pain modulation in human, the communication tried to investigate the effect of intranasal AVP on human headache. The results showed that (1) AVP concentration in both plasma and cerebrospinal fluid (CSF) increased significantly in headache patients, who related with the headache level; (2) there was a positive relationship between plasma and CSF AVP concentration in headache patients; and (3) intranasal AVP could relieve the human headache in a dose-dependent manner. The data suggested that intranasal AVP, which was delivered to the brain through olfactory region, could treat human headache and AVP might be a potential drug of pain relief by intranasal administration.     

[3H]Nitrendipine Binding to Calcium Channels in Bovine and Rat Pituitary

[3H]Nitrendipine was used to label sites in homogenates of bovine anterior and neurointermediate lobes of the pituitary gland. The amount of specific binding in the anterior lobe was 1.82 +/- 0.30 pmol/g wet weight of tissue and the KD was 1.44 +/- 0.02 X 10(-10) M. Preliminary experiments indicated a similar amount of binding in bovine neurointermediate lobe. In competition studies nimodipine and nisoldipine (two potent voltage-sensitive calcium channel blockers) displayed IC50 values of 1.6 and 6.8 X 10(-10) M, respectively. Verapamil and the verapamil-like calcium channel blockers D-600 and tiapamil competed in a complex manner for the [3H]nitrendipine specific binding to bovine anterior pituitary homogenates. Autoradiographical studies demonstrated specific [3H]nitrendipine binding sites distributed approximately equally in the anterior and posterior lobes, but not in the intermediate lobe of the rat pituitary. In general the properties of [3H]nitrendipine binding in the pituitary tissue resemble strongly the properties of [3H]nitrendipine binding in the brain which is believed to be to voltage-sensitive calcium channels. These results provide support for the hypothesis that calcium channels are involved in pituitary hormone secretion and that drugs that interact with calcium channels may modulate the secretory process directly at the level of the pituitary.     

Inhibition of porcine elastase and anhydroelastase by boronic acids

An antiserum against bovine pancreatic trypsin inhibitor (bPTI) has been raised in rabbits. The antiserum does not cross-react with known pituitary hormones and other proteins. A specific radioimmunoassay for bPTI has been developed. The ED50 is about 250 fmol per assay tube and the sensitivity is reliable to 15 fmol per tube. Immunoreactivity could be detected in bovine and sheep pituitaries, bovine brain, and adrenal medulla, but not in human, pig, and rat pituitaries. In the bovine pituitary the immunoreactivity is restricted to the posterior lobe.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

The effect of serotonin agonist 1-(trifluoromethylphenyl)-piperazine on corticotropin releasing factor and arginine vasopressin in rat hypothalamic nuclei

Effects of 1-(m-trifluoromethylphenyl)-piperazine, a serotonin agonist, were examined on rat plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP), and on hypothalamic contents of corticotropin releasing factor (CRF) and AVP, to investigate the role of brain serotonin in ACTH regulation. Both plasma ACTH and AVP levels increased markedly 30 min after injection of the compound and were still elevated at 80 min. CRF and AVP contents in the median eminence decreased 30 min after injection but returned to the basal levels by 80 min. The AVP content in the supraoptic nucleus was elevated 80 min after injection. The CRF and aVP content did not significantly change in the paraventricular, suprachiasmatic and arcuate nuclei. Serotonin or 1-(m-trifluoromethylphenyl)-piperazine did not stimulate the release of ACTH in pituitary cell cultures. These results suggest that both CRF and AVP were secreted into the portal vessels by 1-(m-trifluoromethylphenyl)-piperazine to release ACTH from the anterior pituitary and that both the ACTH and AVP release were stimulated via the brain serotonergic mechanism.     

Effects of prostaglandin E2 and D2 on the release of vasopressin and oxytocin

The effects of prostaglandin (PG) E2 and D2 on the plasma levels of arginine-vasopressin (AVP) and oxytocin (OXT) in rabbits, and on the release of the both hormones from the isolated posterior pituitary of rats were examined. An intraventricular administration of PGE2 to a rabbit raised the plasma levels of the both hormones. An intraventricular injection of PGD2 also increased the plasma level of OXT but not that of AVP. The release of AVP and OXT from fragments of the posterior pituitary of a rat was not influenced by perfusion with Eagle MEM medium containing 10(-6) or 10(-5) M PGE2 and D2.     

Involvement of accessory neurosecretory nuclei of hypothalamus in the formation of hypothalamohypophyseal system during prenatal and postnatal development in rats

We studied the development of direct axonal connections of the accessory neurosecretory hypothalamic nuclei with the posterior pituitary lobe on the fixed rat brain from day 15 of embryogenesis until day 10 of postnatal development using the retrograde diffusion method of the lipophilic fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The marker was applied onto the posterior pituitary lobe and, after incubation in a fixative, fluorescing bodies of nerve cells were visualized in the hypothalamus. Neuronal axons of the retrochiasmatic nucleus were the first of the accessory nuclei to ingrow in the posterior pituitary lobe (on days 16-17 of embryogenesis). Neurons of the circular and dorsolateral nuclei and the nuclei of the median bundle of the forebrain sent their axons to the posterior pituitary lobe starting from the first days of postnatal development. No direct connections of the anterior commissure and perifornical accessory nuclei with the posterior pituitary lobe were found in perinatal development. These facts are discussed in the light of concepts about the different functional role of accessory peptidergic hypothalamic nuclei in rats.     

Angiotensin-converting enzyme (kinase II) in pituitary gland of spontaneously hypertensive rats

Angiotensin-converting enzyme (ACE) (kinase II; dipeptidyl carboxypeptidase, EC 3.4.15.1) activity was measured in pituitary gland of young (4-week-old) and adult (18-week-old) male, spontaneously hypertensive rats (SHR) and in age-matched normotensive male Wistar-Kyoto (WKY) control rats. In the three lobes of the pituitary gland ACE activity was significantly higher in young than in adult animals, in both SH and WKY rats. In the anterior lobe, ACE activity was lower in SHR when compared to age-matched Wistar-Kyoto controls. In contrast, ACE activity in the intermediate lobe of the pituitary gland was higher in SHR, and in particular in young animals. The observed differences between young WKY and SH rats in both the intermediate and anterior lobes did not appear to be due to a modified affinity of ACE for the substrate hippuryl-His-Leu, but to alterations in ACE maximal velocity or number of available molecules. No differences in ACE activity were detected between SHR and WKY rats in the posterior lobe. Total protein content was higher in the intermediate lobe and lower in the posterior lobe of young SHR when compared to normotensive controls. The present results suggest the possibility for a role of pituitary ACE in spontaneous (genetic) hypertension in rats.     

Effect of synthetic atrial natriuretic factor on arginine vasopressin release by the rat hypothalamo-neurohypophysial complex in organ culture

The effect of synthetic rat atrial natriuretic factor (ANF, Arg 101-Tyr 126) was evaluated in an in-vitro model of rat hypothalamo-neurohypophysial complex (HNC) in organ culture in which part of hypothalamus containing a portion of undamaged magnocellular neurons is separated from posterior pituitary by a fluid tight barrier with an intact stalk connecting both structures. ANF, when added to the medium at the hypothalamus site at concentrations of 3 X 10(-5) M to 3 X 10(-7) M, did not change basal AVP release from the posterior pituitary. Similarly, a shorter form of ANF (Cys 105-Tyr 126), reported to be highly potent in inhibiting adenylate cyclase activity in various tissues, exerted no effect on AVP excretion from HNC in organ culture. The application of an hyperosomotic medium (osmolality 324 +/- 2 mOsm/kg H2O) to the hypothalamic side, together with ANF (3 X 10(-6) M), significantly lowered osmotically-stimulated AVP release. It is concluded that ANF has no effect on basal AVP release from HNC in culture and suppresses osmotically-stimulated AVP secretion in this in vitro model.     

Purification and Characterization of a Membrane-Bound Enkephalin-Forming Carboxypeptidase, "Enkephalin Convertase"

Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.     

Hypothalamic action of atrial natriuretic factor to inhibit vasopressin secretion

The ability of synthetic atrial natriuretic factor (ANF) to inhibit vasopressin (AVP) release, as well as its action to inhibit water intake and salt preference in the rat, suggest a role for the peptide in the hypothalamic control of fluid volume in addition to its established actions in the kidney. We report here evidence for a direct, hypothalamic site of action of ANF to inhibit, specifically, AVP secretion. Third cerebroventricular infusion of 1.0 (p less than 0.05) and 2.0 (p less than 0.025) nmoles ANF significantly inhibited AVP release in euvolemic, normally hydrated rats while IV doses of ANF failed to significantly alter AVP release except when 5 nmoles (p less than 0.05) were infused. No significant effects on oxytocin (OT) release were observed. Vasopressin release from median eminence or pituitary, neural lobe explants during static, in vitro incubations was not significantly altered by doses of ANF ranging from 10(-12) to 10(-7) molar. Release of AVP during perifusion of neural lobe explants in the presence of ANF was similarly unaffected. However, AVP and not OT release from hypothalamo-neurohypophysial system explants was significantly inhibited in the presence of 10(-8) and 10(-7) M ANF, suggesting an action of the peptide at the levels of the AVP-producing cell bodies in the included supraoptic nucleus either directly or via an action on an interneuron, and not at the AVP-containing terminal fields in the median eminence or neural lobe.     

Estimation of cholinesterase and choline acetyltransferase in bovine anterior pituitary, posterior pituitary, and pineal body

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were estimated colorimetrically with thiocholine (SCh) esters as substrates in homogenates of bovine anterior pituitary (AP), posterior pituitary (PP), and pineal body (PB), and the levels were referred to those of whole rat brain. The levels of BuChE were very similar in all four tissues, approximately 10.25 μm -BuSCh hydrolysed/g tissue/hr; indicating that this enzymic activity represents a common structural component, perhaps vascular elements. Acetyl-thiocholine (ASCh) hydrolysis by AChE for brain, PP, PB, and AP was 338, 37,24, and 6 μm /g/hr, respectively. Choline acetyltransferase (ChAc) was estimated by the formation of [¹⁴C]acetylcholine from [¹⁴C]acetyl CoA. ChAc activity of posterior pituitary was generally found to be 15–20 per cent that of brain; the activity was always lowest in the anterior pituitary and pineal body, sometimes undetectable, but generally 5–10 per cent that of brain. The basis for the interpretation that cholinergic components in the posterior pituitary are due to acetylcholme-containing nerve endings and in anterior pituitary and pineal body to axons of sympathetic neurons was discussed.     

The immunolocalization of ACTH and alpha MSH in human and rat pituitaries.

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.     

Cholinesterase in the posterior and intermediate lobes of the pituitary

Summary Posterior and intermediate lobes of pituitary glands of cat, rabbit, beef, and rat were examined histochemically for specific (AChE) and non-specific (BuChE) cholinesterase by light and electron microscopy. Acetylthiocholine was utilized in conjunction with ethopropazine to demonstrate AChE, and butyrylthiocholine with BW 284C51 to demonstrate BuChE. Glandular cells of the intermediate lobe of cat, rabbit and rat contained variable amounts of AChE, whereas those of beef contained BuChE. In the posterior pituitary, AChE was detected in the cat, BuChE in the beef and rat, and both AChE and BuChE in the rabbit. In the posterior lobe of all species examined, cholinesterase, whether true or pseudo enzyme, as the case may be, was localized to certain pituicytes and pituicyte-neuron junctions. These histochemical studies failed to identify cholinergic neurons in the posterior pituitary. Large blood vessels of the pituitary were innervated apparently by adrenergic nerves only. Speculations on the role of pituicyte cholinesterase in posterior pituitary secretion are presented.Supported by the Medical Research Council of Canada.Medical Research Associate of the MRC of Canada.     

Schwann cell growth factors.

Purified rat Schwann cells were found to proliferate very slowly in normal growth medium containing 10% fetal calf serum (FCS). Crude extracts of bovine pituitary or brain markedly enhanced Schwann cell growth, while similar extracts of nerve roots, liver and kidney did not. Pituitary extracts were more potent than brain extracts, and extracts from both anterior and posterior pituitary were active. The mitogenic activity of pituitary extracts was reduced by treatment with trypsin, and abolished by pronase and by boiling. A variety of known anterior and posterior pituitary hormones, as well as fibroblast, epidermal and nerve growth factors, were not mitogenic. FCS (greater than 1%) was required for Schwann cell proliferation, but even high concentrations of FCS did not substitute for pituitary or brain extracts, and serum from various other species did not support Schwann cell growth. Although various agents that increase cyclic AMP levels (such as cholera toxin) had been shown to be Schwann cell mitogens, extracts of pituitary or brain did not increase cyclic AMP levels. Extracts of various bovine tissues, including pituitary, brain, liver and kidney, acted synergistically with cholera toxin in stimulating Schwann cell proliferation, although the increase in cyclic AMP induced by the mixture was not greater than that seen with cholera toxin alone. We conclude that there are at least two separate pathways for stimulating Schwann cell division, only one of which involves an increase in intracellular cyclic AMP.