Th. Unger

Enkephalin effects on blood pressure, heart rate, and baroreceptor reflex.

SUMMARY The cardiovascular effects of opioid peptides have been studied. Leudne-enkephalln (Leu- ENK) produced blood pressure (BP) increases following administration into the lateral brain ventricles (i.v.t.), into the cisterns magna (i.e.!.), and following Intravenous (I.Y.) administration. Heart rate (HR) increases were observed following all routes of administration (threshold for BP and HR effects at 03 nmole, maximum at 360 nmoles). The cardiovascular effects were independent of generalized seizures, which may occur at higher doses of enkephaiins (ENK).

Evidence for the presence of enkephalins in the heart

Extracts of guinea pig hearts were subjected to high performance liquid chromatography (HPLC) and the eluted fractions monitored by radioimmunoassays (RIA) for their content of leucine5-enkephalin (Leu-ENK) and methionine5 enkephalin (Met-ENK). Distinct peaks of both Leu-ENK and Met-ENK immunoreactivity were found corresponding to the position of synthetic Leu-ENK and Met-ENK respectively. The ratio of Leu-ENK to Met-ENK content was about 1:4. Chemical sympathectomy with 5-hydroxydopamine (6-OH-DA) produced a dramatic fall in noradrenaline content of the heart by more than 99%, whereas the concentration in Leu-ENK was reduced by only 70%. The Leu-ENK content of the adrenal glands was not affected by this treatment. These observations point to an enkephalinergic innervation of the heart which appears to be mainly of sympathetic origin. The results suggest the participation of enkephalins in cardiac reflex mechanisms.

Effects of lesions in the paraventricular nucleus of the hypothalamus on vasopressin and oxytocin contents in brainstem and spinal cord of rat

The effects of lesions of the paraventricular nucleus in rat hypothalamus (PVN) on the vasopressin (AVP) and oxytocin (OT) contents of the brainstem and spinal cord, as measured by radioimmunoassay, were studied. AVP decreased by 50% and 80% in brainstem and spinal cord of lesioned animals, whereas OT disappeared almost completely. Therefore, in contrast to OT, the PVN is not the only site of origin of AVP-containing nerve fibers projecting to the brainstem.

Components of the Renin-Angiotensin System in the Cerebrospinal Fluid of Rats and Dogs with Special Consideration of the Origin and the Fate of Angiotensin II

From the in vitro and in vivo measurements of the components of the renin-angiotensin system (RAS) in the cerebrospinal fluid (CSF) of rats and dogs, it was concluded that angiotension II (ANG II) is not generated within the CSF in significant amounts, since renin was found to be unmeasurable in CSF under most circumstances. The specific concentrations of angiotensinogen and of converting enzyme (CE) were high. Angiotensin I (ANG I) concentrations were low in CSF, while ANG II levels were comparable to those measured in plasma under control conditions. Neither ANG I nor ANG II penetrated from the blood into the brain ventricles of rats, provided that no unrealistically high doses of ANG II were administered intravenously. This holds true even if high blood pressure increases were induced by intravenous ANG II infusion in deoxycorticosterone acetate (DOCA) and salt-treated rats. However, increased ANG II concentrations were measured in CSF perfusate, when the blood-brain barrier (BBB) was opened by the intracarotid injection of a hyperosmolar urea solution. The brain ventricular perfusion of increasing concentrations of ANG II revealed constant recovery of less than 40%. CSF did not contain angiotensinase activity, but ANG II degradation was high in some periventricular regions. ANG II, the ANG II antagonist saralasin, and the CE inhibitor captopril, respectively, escaped from CSF into circulation when high doses of these substances were applied intraventricularly. We conclude that ANG II in the CSF does not originate from and is not related to plasma ANG II. It is probably not generated within the CSF. ANG II may be synthetized in the brain tissue and be released into the brain ventricles where its rapid degradation occurs in contact with circumventricular structures.

Angiotensin-induced hypertension in the rat. Sympathetic nerve activity and prostaglandins.

To elucidate mechanisms of angiotensin II (Ang II)-related hypertension, we infused angiotensin (76 ng/min s.c.) into rats with minipumps for 10–14 days. Control rats received sham pumps. We measured blood pressure by tail-cuff, and the excretion of aldosterone and prostaglandins (PG) (PGE2, prostacyclin derivative 6kPGF1a, and thromboxane [Tx] derivative TxB2). Angiotensin II increased blood pressure by 20 mm Hg by day 2 and by 90 mm Hg by day 10. Aldosterone excretion increased from 10 to 70 ng/day in Ang II rats by day 7. Urine PGE2 did not increase in angiotensin rats; however, both 6kPGF1a and TxB2 excretion increased with angiotensin. Control rats had no changes in any of these parameters. A sympathetic component was tested in a separate group of angiotensin rats that received phenoxybenzamine (300 μ/kg/day) during angiotensin infusion; their increase in blood pressure of 40 mm Hg at 10 days was less than in those rats with angiotensin alone but more than in control rats. Phenoxybenzamine did not influence the angiotensin-induced increases in excretion of 6kPGFlα or TxB2. Additional groups of conscious angiotensin and control rats were equipped with splanchnic nerve electrodes on day 14 for recording of sympathetic nerve activity. Angiotensin rats had greater basal sympathetic nerve activity than the control rats. Incremental methoxamine injections demonstrated altered baroreceptor reflex function in rats receiving angiotensin. We conclude that increased blood pressure with chronic angiotensin infusion is accompanied by increased production of aldosterone and increased sympathetic tone. The latter may be modulated by PG.

The Renin-Angiotensin-System in the Brain

The biosynthetic pathway and the functions of the hormonal plasma renin angiotensin system are well recognized. Since the components of the RAS were first described within the brain (Ganten et al., Science 1973:64, 1971), evidence has accumulated supporting the existence of a complete RAS endogenous to the brain and playing a role in cardiovascular and volume homeostasis. The high molecular weight peptide precursor angiotensinogen has been cloned and the complete sequence of angiotensinogen is determined. Using cellfree translation techniques it has been demonstrated that the same angiotensinogen molecule is synthesized in the brain and in the liver. The regulation of transcription of the gene, however, appears to be tissue specific. Angiotensin I and the active octapeptide angiotensin II (ANG II) have been characterized in the brain. The distribution of ANG II in the central nervous system has been investigated by chemical as well as by immunohistochemical techniques. The main locations of ANG II are the hypothalamus, the limbic system, the medulla oblongata and the spinal cord; there is good agreement with the localization of ANG II receptors. The key enzymes of the peptide system, i.e. renin, converting enzyme and angiotensinases have equally been shown to be present in the brain and pharmacological inhibition of the synthesizing enzymes leads to reduced angiotensin synthesis and e.g. blood pressure reduction (Ganten et al., Science 221: 869, 1983). Since the genes for the RAS proteins are now known, studies at the cellular and molecular level have become possible. The availability of pharmacological interferences at the sites of peptide generation and peptide action to an extent not equalled for other enzyme peptide systems makes the RAS one of the best studied peptide systems in the brain.

The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and bay K8644 and inhibited by 8-bromo-cyclicGMP

The role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release was studied using the isolated perfused rat heart. The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the protein kinase C pathway, produced a dose-dependent increase in perfusate ANP immunoreactivity. Bay k8644, a putative calcium channel activator, and forskolin, which stimulates adenylate cyclase, induced a sustained increase in ANP secretory rate. TPA in combination with either Bay k8644 or forskolin induced higher ANP secretion than the calculated additive value for each agent. 8-bromo-cyclicGMP and sodium nitroprusside, when given alone, had no effect on ANP secretion, but delayed the TPA-stimulated increase in perfusate ANP. ANP secretion appears therefore to be mediated both by the phosphoinositide and the cAMP system, whereas the cGMP pathway may be inhibitory.

The Intrarenal Renin-Angiotensin-System An Immunocytochemical Study on the Localization of Renin, Angiotensinogen, Converting Enzyme and the Angiotensins in the Kidney of Mouse and Rat*

SummaryThe localization of renin, converting enzyme (CE) and angiotensin II (ANG II) in the kidneys of rats and mice was investigated with immunocytochemical methods. According to the presence and specific intrarenal localization of these components of the renin-angiotensin-system (RAS) our results suggest that in addition to the well known systemic effects of the RAS, there are interactions of its components inside the kidney. These interactions may lead to the generation of an extra portion of ANG II in the renal blood stream with its target cells determined by the localization of CE at the luminal side of well defined endothelial areas. These intrarenal-intravasal reactions may or may not reinforce the action of “systemic” ANG II, generated prerenally. In addition, the existence of true intrarenal-interstitial interactions, with the different components and actions of this intrarenal RAS restricted entirely to the kidney is suggested by our results, particularly the demonstration of ANG II within epitheloid cells and the dissociation of systemic renin and ANG II from their local concentrations in renal hypertensive rats.ZusammenfassungDie intrarenale Verteilung von Renin, Converting enzyme (CE) und Angiotensin II (ANG II) wurde mit immunzytochemischen Methoden an Ratten und Mäusen untersucht. Die hier aufgezeigten spezifischen Verteilungsmuster dieser Komponenten des Renin-Angiotensin-Systems (RAS) legen die Annahme nahe, daß es neben den bekannten systemischen, durch ANG II vermittelten Effekten des RAS auch lokale Interaktionen von RAS-Bestandteilen innerhalb der Niere gibt. — Eine erste Folge dieser Interaktionen dürfte die intrarenale Generation einer zusätzlichen Portion von ANG II im Nierenblutstrom sein, deren Zielgebiet durch die spezifische Lokalisation von CE in bestimmten Endothelbereichen der Nierenstrombahn bestimmt wird. Solche intrarenal-intravasalen Reaktionen können für sich wirksam werden, aber auch den Effekt von „systemisch“, d.h. prärenal generiertem ANG II verstärken. — Unsere Ergebnisse sprechen weiter dafür, daß es neben diesen intrarenal-intravasalen auch echte intrarenal-interstitielle Interaktionen der RAS-Komponenten gibt, deren Wirkung sich über das im Interstitium der Nierenrinde generierte ANG II allein auf die Niere beschränkt. Für das Vorhandensein eines solchen lokal-intrarenalen RAS spricht vor allem der Nachweis von ANG II in den epitheloiden Zellen des JGA und die Dissoziation des systemischen — an der Plasmakonzentration abzulesenden — Renin und ANG II von deren lokal-intrarenalen Konzentrationen bei renal hypertensiven Ratten.

Angiotensin stimulates oxytocin release.

Abstract A sensitive and specific radioimmunoassay for oxytocin (OT) was developed to study the effect of angiotensin II (ANG II) upon neurohypophyseal OT release in conscious rats. ANG II injected into the lateral cerebral ventricle (i.c.v.) produced within 60 seconds a steep increase in plasma OT concentration from a control value of 10.46 ± 1.35 fmol/ml to 88.95 ± 5.06 fmol/ml and 119.56 ± 11.46 fmol/ml following 10 and 100 ng i.c.v. ANG II, respectively. Inhibition of OT release by simultaneous application of the ANG II antagonist {Sar 1 , Ile 8 }-ANG II suggests that ANG II acted via specific angiotensin receptors in the brain.

Converting-enzyme in the choroid plexus, brain, and kidney: Immunocytochemical and biochemical studies in rats

In rats, immunoreactivity was demonstrated in the brush border of the choroid plexus, in the wall of blood vessels in the brain, and in the brush border of the proximal tubules of the kidney, using the peroxidase-antiperoxidase technique (PAP) and an anti-pulmonary converting-enzyme antibody. These findings correlate with biochemical data of converting-enzyme activity in the choroid plexus and in other tissues. Possible functions of the enzyme in relation to its localization are discussed.