Rat medial basal hypothalami (MBH) or neurointermediate lobes of the hypophysis (NIL) were superfused in vitro and stimulated electrically. The evoked release of vasopressin from the MBH was enhanced to 700% of controls when the tissue was taken from adrenalectomized rats. By contrast, the evoked release of vasopressin from the NIL was not changed after adrenalectomy. After bilateral lesions of the paraventricular nuclei, the evoked release of vasopressin from the MBH was reduced in subsequent in vitro experiments. The marked changes in vasopressin release occurred in spite of no or only small changes in the total tissue content of vasopressin. These data support the view that vasopressin may be released from the external layer of the median eminence into the hypophysial portal blood after activation of the hypothalamo-pituitary-adrenal axis.

Naloxone increases the electrically induced vasopressin release from rat pituitary neurointermediate lobes under appropriate stimulation conditions. In order to examine a possible role of hypophyseal opioid peptides we studied in vitro the effect of opioid peptides and of naloxone on the electrically induced vasopressin release from the rat neurointermediate lobe or isolated neural lobe of the hypophysis. (D-Ala2,D-Leu5)-enkephalin (5 microM), dynorphin-(1-13) (Dyn; 0.2 microM), beta-endorphin (beta-End; 0.02 and 0.2 microM) and also naloxone (1 or 10 microM) increased the evoked vasopressin release from the neurointermediate lobe, but in higher concentrations (2 microM) Dyn or beta-End had no effect. After removal of the intermediate lobe, beta-End 2 microM inhibited, while naloxone 10 microM did not change the evoked vasopressin release from the isolated neural lobe. These results demonstrate that hypophyseal opioid peptides can influence vasopressin release in several ways and suggest that endogenous opioids predominantly provide inhibitory influences which depend on the presence of the intermediate lobe.

Rat posterior pituitaries were superfused in vitro and stimulated electrically. The concentrations of vasopressin in the superfusion medium were determined by radioimmunoassay. When the pulses were applied in 10 sec trains with 10 sec intervals, vasopressin release per pulse increased progressively over the frequency range of 3‐12 pulses/sec applied within the trains. The release was blocked by addition of tetrodotoxin or by removal of calcium ions from the superfusion medium. The opiate antagonist naloxone 1 or 10 microM was introduced into the superfusion medium before a second period of stimulation and enhanced vasopressin release from neurointermediate lobes after phasic stimulation at 9 pulses/sec within the trains, when compared to controls. However, naloxone 10 microM had not effect on vasopressin release from isolated neural lobes (intermediate lobes removed), although the addition of camel beta‐endorphin 2 microM inhibited vasopressin release in a naloxone‐reversible manner. After continuous stimulation at a frequency of 13 Hz naloxone 10 microM did not influence the release of vasopressin from neurointermediate lobes. We conclude that the evoked release of vasopressin from the neurointermediate lobe is reduced by an endogenous opiate of intermediate lobe origin, possibly beta‐endorphin. Appropriate stimulation conditions are necessary for this mechanism to function.

Studies on the cholecystokinin-like immunoreactivity (CCK-IR), of the medial basal hypothalamus (MBH) were performed in rats. In immunohistochemical studies, a dense CCK-IR-positive staining was found in the external layer of the median eminence adjacent to portal capillaries. Either high potassium (56 mM) or veratridine (1-50 micrograms/ml) stimulated the release of CCK-IR from MBH incubated in vitro. This increase in CCK-IR release depended on the presence of calcium. In vivo, the content of CCK-IR in the MBH was reduced after bilateral adrenalectomy and this effect of adrenalectomy was reversed by dexamethasone treatment. The content of CCK-IR in the neurointermediate lobe of the pituitary gland was not changed under these conditions. These results raise the possibility that after activation of the hypothalamo-pituitary-adrenal axis cholecystokinin may be secreted from nerve terminals in the external layer of the median eminence into the hypophyseal portal blood.

Transection of subfornical organ efferents in the rat prevented the vasopressin release in response to intravenous angiotensin II infusion or following a small dose of the β-sympathomimetic amine isoprenaline (30 μ/kg i.m.). In contrast, this lesion had no effect on vasopressin release after hypertonic saline injection or a high dose of isoprenaline (480 μg/kg i.m.). We conclude that blood-borne angiotensin II induces vasopressin release by acting on the subfornical organ; depending on the dose of isoprenaline, activation of the endogenous renin-angiotensin system may mediate isoprenaline-induced vasopressin release.

Blood-borne angiotensin II induces release of β-endorphin-like immunoreactivity (β-EI) from rat anterior pituitary gland. To study the mechanism of action we investigated in rats the effect of transection of subfornical organ efferent projections on angiotensin-induced β-EI release in vivo and also the direct action of angiotensin II on β-EI release from isolated adenohypophyses in vitro. (i) No effect of transection of subfornical organ efferents on the increase in plasma β-EI following intravenous infusions of angiotensin II was found. (ii) When anterior pituitary quarters were continuously superfused in vitro, angiotensin II (1 – 10 nM) caused release of β-EI into the superfusion medium in a dose-dependent manner. The stimulatory effect of angiotensin II (3 nM) was blocked by the receptor antagonist saralasin (300 nM). We conclude that β-endorphin release by blood-borne angiotensin II, in contrast to other central effects of angiotensin, is not mediated by the subfornical organ; instead a direct action of angiotensin II on the adenohypophysis could be a mechanism of action responsible.

Rat medial basal hypothalami were superfused in vitro. The effect of angiotensin II on vasopressin outflow was investigated. Angiotensin II (10 nM or 1 microM, added to the superfusion medium) increased the veratridine-evoked vasopressin release. The higher concentration also slightly elevated the basal outflow. The effect of angiotensin II was blocked by saralasin. We conclude that angiotensin II can act on the median eminence and/or on the stump of the pituitary stalk to promote the release of vasopressin, which then may influence anterior pituitary hormone secretion.

The effect of cholecystokinin on the release of β-endorphin immunoreactivity from anterior pituitary quarters and from cultures of dispersed anterior pituitary quarters was investigated. Cholecystokinin (> 10−7 M) caused a slight release of β-endorphin immunoreactivity from the pituitary quarters only.

Lesion of the fibers of the corpus callosum in the midline causes a bilateral increase in the concentration of cholecystokinin-like immunoreactivity in some cortical areas in rat brain, i.e. areas frontopolaris and postcentralis caudalis. Both these areas are homeotopically connected by callosal fibers.

Large Amounts of cholecystokinin-octapeptide (CCK) are present in the rat caudatoputamen. The peptide occurs in axons and nerve endings but not in perikarya. The origin of CCK in the caudatoputamen was investigated with the use of immunocytochemistry and a radioimmunoassay specific for CCK. Although a small amount of CCK (approximately 30 percent) originates in the amygdaloid complex, the bulk of the peptide (approximately 70 percent) occurs in processes of neurons located ventral to the caudatoputamen, that is, the claustrum or the piriform cortex. The claustrum and piriform cortex receive inputs from various cortical areas and the olfactory system, respectively, and may process information and relay it to the caudatoputamen. Thus CCK may by the transmitter in the final common pathway linking various cortical areas and the olfactory system to the caudatoputamen.

The regional distribution of cholecystokinin (CCK) in the rat brain was determined utilizing a radioimmunoassay which detects both gastrin and CCK. CCK concentration is highest in the caudate nucleus (10–14 ng CCK8 equivalents/mg protein), followed by the cerebral cortex. Within the cerebral cortex, CCK is highest in the cingulate, pyriform, and entorhinal areas. There are substantial CCK concentrations in all other brain regions except pons, medulla and cerebellum. CCK is widely distributed in the hypothalamus, where it is highest in the median eminence and ventromedial nucleus. Considerable CCK-like immunoreactivity is also present in the posterior lobe of the pituitary gland, but is not detectable in anterior and intermediate lobes.

The cortical information flow via the perforant path represents a major excitatory projection to the hippocampus. Lesioning this projection leads to massive degeneration and subsequently to reorganization in its termination zones as well as in primary non-affected subfields of the hippocampus. The molecular mechanisms and factors which are involved in the postlesional events are poorly defined. Using a differential display reverse transcription–polymerase chain reaction (DDRT-PCR) strategy, we located one band which occurred only in control hippocampus lanes and almost disappeared in the lanes of lesioned hippocampi. By sequencing, we identified the corresponding gene as cholecystokinin (CCK). Northern blot analysis confirmed a decreased transcription of CCK after lesion. In situ hybridization analysis was performed for localization and quantification of altered CCK transcription. We noted a significant downregulation of CCK transcription in the hippocampus (20%) and in the contralateral cortex (12%) 1-day after lesion (dal) and an increased signal in the ipsilateral cortex (10.5%). This pattern was altered, showing upregulation of CCK mRNA expression, reaching its highest level of 70% above control levels at 5 dal. In the hippocampus, the control level was reached again at 21 dal, whereas the cortex reached the control level at 10 dal. In comparison, the mRNA transcripts of the receptors CCKA and CCKB remained unchanged. Since CCK-containing neurons are involved in the modulation of pyramidal and granule cell excitability, our data indicate a time course correlation between CCK mRNA expression and postlesional axonal sprouting response in the hippocampus.

Summary We examined whether the activation of the renin -angiotensin system after intramuscular isoprenaline injection contributes to the simultaneous increase in vasopressin release. Plasma concentrations of vasopressin and angiotensin II were measured in conscious rats using specific radioimmunoassays. Intravenous infusions of angiotensin II caused a dose-dependent increase in vasopressin release. Intravenous infusions of the angiotensin II antagonist saralasin did not diminish the isoprenaline-induced vasopressin release. However, the curve relating the isoprenaline-induced decrease in blood pressure and the concomitant increase in vasopressin levels was shifted to the right and no longer linear in saralasin-treated rats. Nephrectomy diminished the vasopressin release caused by isoprenaline when compared to sham-operation. The correlation between the decrease in blood pressure and the simultaneous vasopressin release was changed in a strikingly similar manner by nephrectomy and by saralasin infusions. It may be concluded that small doses of isoprenaline, which cause only minor decreases in blood pressure, induce vasopressin release via the renin-angiotensin system. However, the contribution of this system to vasopressin release declines as hypotension becomes more severe.

The concentration of choline acetyltransferase, a specific marker for cholinergic neurons, was determined in the supraoptic nucleus after a variety of lesions. Surgical lesions immediately rostral as well as medial and lateral to the nucleus did not affect the concentration of the enzyme. Only lesions which separated the nucleus from the posterior part of the lateral hypothalamus slightly decreased its concentration in choline acetyltransferase. It is concluded that the bulk of the cholinergic neurons is in the supraoptic nucleus or its immediate vicinity.

The localization of the GABAergic neurons which send efferent fibers to the supraoptic nucleus was investigated. For this purpose the activity of glutamic acid decar☐ylase, a specific marker for GABAergic neurons, was determined in the supraoptic nucleus after a variety of lesions. The severance of fibers from the mesencephalon and the mediobasal hypothalamus, as well as from the hippocampus, had no effect. However, lesions rostral to the nucleus reduced its activity in glutamic acid decar☐ylase by about 40%, as did the infusion of kainic acid into the nucleus accumbens.

The effects of intravenous infusion of angiotensin I, II and (des‐1‐Asp) angiotensin II (angiotensin III) on the plasma vasopressin levels, with and without converting enzyme inhibition, were investigated in conscious rats by use of a specific radioimmunoassay. All three peptides caused a dose‐dependent increase in vasopressin release, angiotensin III infusion being less effective than angiotensin I or II. The converting enzyme inhibitor, SQ 20881 (Pyr‐Trp‐Pro‐Arg‐Pro‐Gln‐Ile‐Pro‐Pro) (1.0 mg/kg, i.v.), had no effect on the plasma vasopressin concentrations in bilaterally nephrectomized rats, but increased them in intact or sham‐operated animals. SQ 20881 potentiated vasopressin release elicited by angiotensin I, leaving that elicited by angiotensin II unchanged. The receptor antagonist, saralasin, prevented the angiotensin‐induced increase in plasma vasopressin concentration, even after pretreatment with SQ 20881. These data support the assumption that the renin‐angiotensin system may be involved in the control of vasopressin release, and indicate that in addition to angiotensin II, angiotensin I and III may also contribute, acting in concert.

Bilateral adrenalectomy combined with a sodium-deficient diet caused a time-dependent increase in plasma renin concentration in rats. Seventy-two hours after adrenalectomy the inhibitors of prostaglandin biosynthesis indomethacin and meclofenamate diminished the plasma renin concentration by about 50%. This effect was independent of the amount of renin released. Indomethacin and meclofenamate fully retained their ability to reduce the plasma renin concentration when the renal sympathetic nerves or the macula densa cells of the kidneys no longer contributed to renin release. It is concluded that in adrenalectomized rats renal prostaglandins affect the baroreceptor-mechanism in the afferent arterioles and thus enhance renin release.

1. A new column-chromatographic method is described for the simple and reproducible determination of the concentration of [des-Asp1]angiotensin II (angiotensin III) in rat plasma. 2. The method uses the different abilities of the ion-exchange resins Dowex 1 (X8) and Bio Rex 70 to bind angiotensin II and angiotensin III. Under the conditions used, Bio Rex 70 binds only angiotensin III. Angiotensin II and its hexapeptide metabolite [des-Asp1,des-Arg2]angiotensin II pass the resin with the effluent. Dowex 1 (X8) binds angiotensin II and the hexapeptide metabolite, whereas it does not extract angiotensin III. It does not separate angiotensin II from the hexapeptide. Therefore the sum of both peptides is expressed as angiotensin II-like activity. The ratio of the concentrations, angiotensin II/hexapeptide, was 5:1. 3. In normal rats the plasma concentration of angiotensin III was 20 fmol/ml (SD 15; n = 8), and angiotensin II-like activity was 60 fmol/ml (SD 35; n = 8). 4. The beta-sympathomimetic amine isoprenaline caused a time- and dose-dependent increase in plasma angiotensin III and angiotensin II-like activities. 5. Under the conditions studied angiotensin III contributed approximately 25% to the total amount of angiotensins in plasma.