Lanny C. Keil

Angiotensin II immunohistochemistry of the rat brain.

Immunoreactive angiotensin II (AII) in rat brain is identified histochemically by the unlabelled antibody-enzyme method. Addition of protease inhibitors, phenylmethyl sulfonyl fluoride (PMSF) and ethylenediamine tetra-acetic acid (EDTA), to subzero (-10 degrees C) fixation with propylene glycol and formaldehyde provides reproducible preservation of immunoreactive AII. Synaptic boutons within the brainstem and cerebellum contain immunoreactive AII. The deep cerebellar nuclei have the richest density of AII positive synapses. Other nonneuronal cells such as pinealocytes, pituicytes, and 3rd ventricular tanycytes contain immunoreactive AII. This dual AII localization suggests that both neurons and blood vessels may be involved in the transport of AII to sensitive sites within the rat brain.

Effect of Chronic Crowding and Cold on the Pituitary-Adrenal System: Responsiveness to an Acute Stimulus during Chronic Stress

The effects of chronic exposure to crowding or cold stress were assessed in male rats over a period of 8 weeks. Body growth was inhibited by both conditions. Anterior pituitary weights decreased after 2 weeks of either treatment, but by 8 weeks the glands of cold exposed rats had hypertrophied. Cold resulted in a sustained adrenal and testicular hypertrophy, whereas crowding had no such effect. Plasma and adrenal corticosterone levels were elevated after 1 week of cold or crowding, followed by a marked reduction after 8 weeks in cold-exposed animals only. Plasma ACTH remained unchanged until 8 weeks when it started rising in both groups of animals. The data obtained in these experiments indicate that the pituitary-adrenal system responds to chronic stress neither by stabilization at a new steady state nor by a return to a period of normalcy, at least within the 8 weeks of exposure studied. The responsiveness of these chronically stressed animals to an additional acute stressful stimulus (1 min of ether) was also evaluated. Crowding depressed both the plasma and adrenal corticosterone response to ether anesthesia at 1 week. Cold exposure enhanced the plasma corticosterone response at all intervals (1–8 weeks) studied and enhanced the ACTH response at 1 and 2 weeks.

Effects of centrally administered endogenous opioid peptides on drinking behavior, increased plasma vasopressin concentration and pressor response to hypertonic sodium chloride

Intracerebroventricular (i.v.t.) administration of beta-endorphin or leucine5-enkephalin inhibited drinking behavior, the pressor response and increased plasma vasopressin concentration stimulated by an acute elevation in CSF sodium chloride concentration (10 microliter, 1 M NaCl i.v.t.). These effects of endogenous opioid peptides were prevented by naloxone, indicating opiate receptors were required for the biologic response. Drinking behavior associated with regulatory stimuli operant during dehydration was also inhibited by opioid peptides. beta-Endorphin (i.v.t.) delayed the onset and/or reduced the volume of water consumed in response to hypertonic sodium chloride (relative cellular dehydration), polyethylene glycol (hypovolemia) and food-associated drinking behavior. Inhibition of drinking did not appear related to sensory-motor dysfunction as another motivated behavior, eating (onset, amount consumed) was unaffected by beta-endorphin. It is concluded from these results that centrally administered endogenous opioid peptides inhibit sodium chloride-stimulated cerebral mechanisms affecting blood pressure and hydration.

Enkephalin inhibition of angiotensin-stimulated release of oxytocin and vasopressin

The effect of leucine5 -enkephalin on angiotensin II (AII)-stimulated release of oxytocin and vasopressin (VP) was investigated in the conscious male rat. Changes in the plasma concentration ([]) of both oxytocin and VP were measured in animals: (1) 60 s after intracerebroventricular (i.v.t.) administration of either artificial cerebrospinal fluid (CSF) or CSF with AII (10, 50, 100 ng/5 microliter); (2) 30, 60, 90 and 300 s after single injection of AII (50 ng/5 microliter; i.v.t.) or CSF and (3) 60 s after AII (50 ng/5 microliter) or CSF in animals pretreated with leucine5 -enkephalin (100 ng/5 microliter; i.v.t.) or CSF (5 microliter). Oxytocin and VP were quantified by radioimmunoassay and values corrected for 100% recovery. The development of a sensitive radioimmunoassay for oxytocin is described. The antiserum for oxytocin enabled detection of greater than or equal to 0.8 pg/ml oxytocin with cross-reactivity of 0.01% with arginine vasopressin and 0.10% with arginine vasotocin. The inter- and intra-assay coefficients of variation were 3-9% and 3.2%, respectively. The hypotheses being tested were that i.v.t. injection of AII stimulates release of both neurohypophysial hormones non-selectively and that leucine5 -enkephalin inhibits both AII-stimulated oxytocin and VP release. Angiotensin II at doses ranging from 10 to 100 ng/5 microliter, i.v.t. increased the plasma concentration of both oxytocin and VP. Plasma levels of both neurohypophysial hormones were elevated 30 s after AII administration i.v.t. and remained elevated 300 sec later.(ABSTRACT TRUNCATED AT 250 WORDS)

The Organum Vasculosum Laminae Terminalis: A Critical Area for Osmoreception

Publisher Summary The volume and composition of the body fluids is maintained by coordinated control of water intake by thirst mechanisms and water output via vasopressin secretion. This chapter describes and presents evidence in favor of the organum vasculosum laminae terminalis (OVLT) as site of osmoreception (both osmotic and volaemic regulation of water balance). Evidence presented in the chapter demonstrates that the osmoreceptors behave as if they were located in an area of the brain lacking the blood-brain barrier and that this area is likely to be in the anterior hypothalamus. Although, there is electrophysiological evidence that cells elsewhere in the brain, such as the supraoptic nucleus, show osmosensitivity, their capacity to respond to reasonable osmotic challenges is in question. Animals with discrete lesions of the OVLT have greatly diminished responses to osmotic stimuli and abolition of these responses over reasonable physiological range, whereas the identity of the osmoreceptors remains elusive; their anatomical home, the OVLT, is more certain.

Localization of angiotensin in rat brain.

cytes, posterior pituicytes, third ventricular tanycytes, and some cells within the spinal trigeminal tract. Preincubating the All antiserum with excess angiotensin I and antidiuretic hormone did not affect this distribution while pre-incubation with All abolished it. Cross-reactivity of All antiserum with angiotensin I, dog renin substrate, and synthetic renin substrate, tetradecapeptide was less than 1%. However, the antiserum showed 70% cross reactivity with angiotensin III (Al!!); therefore this immunohistochemical map could also correlate with the presence of Al!!. This morphology of the All/All! localization in the rat brain when correlated to the known patterns of All/All! receptors within brain suggests angiotensin may be transported to sensitive brain sites by both neurons and vascular channels.

Urethane Anesthesia in Rats

Anesthesia in rats produced by urethane administered intraperitoneally caused (1) peritoneal fluid accumulation; (2) inability to undergo a renal response to NaCl or water loading, and (3) pronounced

Identification of vasopressin in the subfornical organ region: effects of dehydration

The subfornical organ (SFO), a neuroendocrine structure implicated in saltwater homeostasis, contains secretory structures histochemically similar to those in the neurohypophysis. Because of these morphological similarities, we compared levels of vasopressin (vp) in the SFO area and the adjacent hippocampal commissure-fornix (HC-F) of normally hydrated and 48 h water-deprived (WD) rats. VP in the SFO region from normally hydrated rats was 44 +/- 5 pg/mg protein, 3.5 +/- 0.4 ng/g wet weight or 3.6 +/- 0.4 pg/SFO. These concentrations increased (P less than 0.05) about twofold after WD. The content of VP, ng/g wet weight, in HC-F was higher (P less than 0.05) than the SFO area and also increased (P less than 0.05) after WD. VP was detected in other fiber tracts, anterior commissure (AC) and fornix (F), but was unchanged by WD. Changes in hormone observed in the SFO and HC-F regions were therefore not part of a generalized increase of VP in the brain, nor can they be ascribed to elevated plasma levels. Thus, VP changes in the SFO region may be functionally significant and related to an SFO endocrine role in hydration. VP in fiber tracts (F, AC) unassociated with the hypothalamo-hypophysial system and unchanged after WD may suggest an unidentified role of this hormone in the central nervous system.

Effect of Chronic Intracerebroventricular Angiotensin II Infusion on Vasopressin Release in Rats

Angiotensin II (AII) was infused into the lateral cerebral ventricle of rats given water, isotonic saline, or hypertonic saline ad libitum, or 40 ml water/day. Fluid intake, change in body weight, plasma [Na+], and plasma and pituitary arginine vasopressin (AVP) levels were measure. Isotonic saline or AII (1 micrograms/microliter saline) was infused at 1 microliter/h for 5 days using osmotic minipumps. AII increased fluid intake of rats given isotonic saline to drink; they consumed an average of 269 +/- 25 ml/day on day 5. AII infusions in rat given water or isotonic saline to drink decreased plasma [Na+] with no changes in plasma or pituitary AVP. However, in rats given hypertonic saline, plasma [Na+] remained at control levels while plasma AVP increased. In water-restricted rats, the effects of AII were intermediate: a small decrease in plasma [Na+] and a small increase in plasma AVP. From these results, it is suggested that although acute AII administration elicits AVP release, this effect diminishes during chronic AII infusion, coincident with reduced plasma [Na+].

Arterial baroreceptors control blood pressure and vasopressin responses to hemorrhage in conscious dogs

The goal of this study was to determine the role of arterial baroreceptors in the reflex control of arginine vasopressin (AVP), renin, and cortisol secretion in response to a 30-ml/kg hemorrhage in conscious dogs. The hormonal responses were measured in six dogs under four treatment conditions: 1) intact, 2) acute cardiac denervation (CD) by intrapericardial infusion of procaine, 3) after sinoaortic denervation (SAD), and 4) during combined SAD + CD. In the intact condition, mean arterial pressure (MAP) was maintained at control levels until blood loss reached 20 ml/kg and the absolute magnitude of the fall at 30 ml/kg was 35 ± 10 mmHg. Similar responses were obtained during acute CD. In contrast, MAP fell earlier (at 5 ml/kg, P < 0.05) and to much lower levels in both the SAD and SAD + CD conditions. The individual slopes relating systolic pressure to plasma AVP, renin activity (PRA), and cortisol were used to compare the treatment effects using a 2 × 2 factorial analysis. There was a significant ( P < 0.01) effect of SAD on the slope relating AVP to systolic pressure but no effect of CD and no SAD × CD interaction. In contrast, there was no effect of either SAD or CD on the relationship between PRA or plasma cortisol and systolic pressure. These results indicate that maintenance of blood pressure and the normal pattern of AVP secretion during hemorrhage depend on intact arterial baroreceptor reflexes.The goal of this study was to determine the role of arterial baroreceptors in the reflex control of arginine vasopressin (AVP), renin, and cortisol secretion in response to a 30-ml/kg hemorrhage in conscious dogs. The hormonal responses were measured in six dogs under four treatment conditions: 1) intact, 2) acute cardiac denervation (CD) by intrapericardial infusion of procaine, 3) after sinoaortic denervation (SAD), and 4) during combined SAD + CD. In the intact condition, mean arterial pressure (MAP) was maintained at control levels until blood loss reached 20 ml/kg and the absolute magnitude of the fall at 30 ml/kg was 35 +/- 10 mmHg. Similar responses were obtained during acute CD. In contrast, MAP fell earlier (at 5 ml/kg, P < 0.05) and to much lower levels in both the SAD and SAD + CD conditions. The individual slopes relating systolic pressure to plasma AVP, renin activity (PRA), and cortisol were used to compare the treatment effects using a 2 x 2 factorial analysis. There was a significant (P < 0.01) effect of SAD on the slope relating AVP to systolic pressure but no effect of CD and no SAD x CD interaction. In contrast, there was no effect of either SAD or CD on the relationship between PRA or plasma cortisol and systolic pressure. These results indicate that maintenance of blood pressure and the normal pattern of AVP secretion during hemorrhage depend on intact arterial baroreceptor reflexes.