Raymond H. Abhold

Enkephalin release into the ventral tegmental area in response to stress: modulation of mesocorticolimbic dopamine

Enkephalin-containing neuronal fibers and perikarya, and opioid receptors are present in the A10 dopamine (DA) region, and many studies have implicated enkephalin as a neuromodulator of A10 DA neurons projecting to the prefrontal cortex and certain limbic nuclei. Footshock stress is known to activate the A10 DA neurons projecting to the prefrontal cortex and nucleus accumbens, and the present study was designed to evaluate the possibility that footshock-induced release of enkephalin into the A10 DA region may play a role in activating the DA neurons. Microinjection of the quaternary opioid antagonist, naltrexone methobromide (NMB), into the ventral tegmental area (VTA; subnucleus of the A10 DA region) significantly attenuated the increase in DA metabolism produced by exposure to footshock (0.2 mA; 200 ms on; 800 ms off for 20 min) in the prefrontal cortex and nucleus accumbens. Rats were exposed to footshock for 5, 10 or 20 min and a time-dependent decrease in the level of immunoreactive Met-enkephalin was measured in the midline A10 region, but not in the lateral A10 region. It has been shown that daily exposure to footshock enhances the motor stimulant effect of intra-VTA injection of the enkephalin analogue, [D-Ala2,Met]-enkephalinamide (DALA). Rats were pretreated with an intra-VTA injection of NMB prior to daily exposure to footshock, and it was found that NMB abolished the potentiating effect of daily footshock on subsequent intra-VTA injection of DALA. Taken together, these data indicate that footshock stress enhances the release of enkephalin into the A10 region, and that this enkephalin activates A10 DA neurons projecting to the prefrontal cortex and nucleus accumbens.

Somatodendritic release of endogenous dopamine: in vivo dialysis in the A10 dopamine region

Using in vivo dialysis in the A10 region of the anesthetized rat, somatodendritic release of endogenous dopamine was demonstrated. Although endogenous dopamine release from the A10 region was enhanced by amphetamine pretreatment in a dose-related manner, the amount of dopamine released was markedly less than the axonal release of dopamine measured simultaneously in the nucleus accumbens.

Elevations in plasma angiotensin II with prolonged ethanol treatment in rats

Chronic alcohol consumption frequently leads to hypertension in humans. While previous reports have implicated the renin-angiotensin system as a potential mediator of this effect, plasma angiotensin II (AII) levels were either not measured or yielded negative results. The present investigation noted significant elevations in circulating AII in rats intubated daily with ethanol (4 g/kg) for 50 days. Animals administered ethanol only once evidenced AII concentrations equivalent with water intubated controls. Radioligand binding assay data indicated no differences in the number or affinity of Sar1,Ile8-AII binding sites in the thalamus, septum-anterior ventral third ventrical region or adrenal gland comparing those groups chronically treated with ethanol to water intubated controls. These results may support a role for the vasoconstrictive hormone AII in the etiology of alcohol-induced hypertension.

Characterization of angiotensin binding to gerbil brain membranes using [125I]angiotensin III as the radioligand

The observation that an Angiotensin II (AII)-sensitive species, the gerbil, exhibited little or no [125I]AII binding to brain membranes led to the hypothesis that AIIs central action may be mediated by smaller and/or modified fragments of AII. This possibility was assessed, in part, by examining the ability of gerbil brain membranes to specifically bind [125I]desAsp1 AII (AIII), a heptapeptide fragment of AII. Specific binding was evident throughout the gerbil brain with highest binding in the septum (containing the subfornical organ), anterior ventral third ventricular region, hypothalamus (containing the median eminence), and striatum. This binding was found to possess many of the properties commonly associated with binding to membrane bound receptors. The binding within the circumventricular organs had characteristics that set them apart from the other central nervous tissues examined. Both the olfactory bulb and adrenal gland appeared to have two different angiotensin binding sites. It appears that the binding sites within the brain interact with a product of the metabolism of AIII or AII rather than the peptides themselves. The results suggest that [125I]AIII appears to be a better ligand than [125I]AII in the binding assay because it is more readily degraded to another substance.

Improved immunohistochemical staining of angiotensin II in rat brain using affinity purified antibodies.

Recent immunohistochemical studies that have sought to detect angiotensin II/III (AII/AIII) immunoreactive material in the brain have been forced to rely on a small number of antisera because most AII/AIII antibodies have unexplainably proved unsuitable for immunohistochemistry. Although extremely useful tools, these antisera have suffered from high background staining. The purpose of this study was to re-examine and characterize the staining using the most popular AII/AIII antiserum (Denise) before and after purification on an AII CH-sepharose affinity column. The use of crude AII/AIII antiserum resulted in the staining of large varicosities and cell bodies. Fibres were all but invisible owing to extensive background staining. In contrast, the purified antibodies yielded little background staining and produced a discrete staining of AII/AIII fibres with small varicosities in the paraventricular-hypophysial pathway and of cell bodies of large hypothalamic neurones. In addition punctate staining demarcated the perikarya of some neurones and resembled boutons containing immunoreactive AII/AIII. Biochemical and histochemical analysis of the crude antiserum, the affinity purified antibodies and other fractions off the sepharose column demonstrated that a large portion of the total staining (various types of background) seen with crude antiserum and column fractions was not to AII/AIII or several angiotensin-derived fragments. Furthermore, successful preabsorption blanks for the purified antibodies could only be achieved with AII coupled through its N-terminal, suggesting that these purified antibodies reacted best with conjugated angiotensin in the fixed tissue. In total the results of this study indicate that the background staining seen with crude antiserum is not to AII/AIII. The use of affinity purified antibodies greatly enhances resolution, enabling one to visualise even small fibres in rats not treated with colchicine, and should improve our ability to develop accurate maps of central angiotensinergic pathways.

Characterization of angiotensin binding in the African Green monkey

The observation that there are differences in the characteristics and distribution of angiotensin receptors in the central nervous system of mammalian species led to the analysis of angiotensin binding in a primate model, the African Green monkey. Initial studies using [125I]angiotensin II ([125I]AII) as the radioligand showed binding in peripheral tissues but little binding in the central nervous system. Conversely, binding studies using [125I]AIII as the radioligand indicated more central nervous binding with diminished peripheral binding. Specific binding of [125I]AIII is evident throughout the brain with high binding in the circumventricular organs, striatum, caudate nucleus, olfactory bulb and localized areas of the thalamus and cerebral cortex. This binding was found to possess many of the properties commonly associated with binding to membrane-bound receptors. The specifically bound radioligand extracted from incubations of [125I]AIII and central nervous tissue appears to be a product of the metabolism of [125I]AIII rather than the peptide itself. Binding of [125I]AII does occur in peripheral tissues and to a limited extent in the cerebellum, but to a different receptor from that characterized using [125I]AIII. These results are similar to those seen in the gerbil and raise questions concerning the utilization of the rat as the primary model for studying the biochemistry of the brain-angiotensin system in humans.

Comparison of 125I‐Angiotensin III and 125I‐Angiotensin II Binding to Rat Brain Membranes

The binding of 125I‐angiotensin III (125I‐ANG III) to rat brain membranes was examined and compared with that of 125I‐angiotensin II (125I‐ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I‐ANG III‐binding sites were observed based on affinity (KD= 0.13, 1.83, and 10.16 nM) and capacity (Bmax= 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I‐ANG II‐binding sites of high affinity (KD= 0.11 and 1.76 nM) and low capacity (Bmax= 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross‐displacement studies confirmed that the two highest‐affinity 125I‐ANG Ill‐binding sites and the 125I‐ANG II‐binding sites were the same. On the other hand, the binding of 125I‐ANG III to the low‐affinity 125I‐ANG III‐binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these pep‐tides to central angiotensin receptors.

In Vitro Down Regulation and Possible Internalization of Central Angiotensin Receptors

Angiotensin receptors in peripheral tissues have been shown to up or down regulate in response to changes in circulating angiotensin2/3. Nevertheless it has been difficult to demonstrate changes in central angiotensin receptors in response to alterations in blood or cerebral spinal fluid (CSF) levels of angiotensins4/5. Failure to detect such changes could be the result of a dilution effect whereby only a small percentage of angiotensin receptors are exposed to altered ligand levels or a rapidly recycling population of receptors. This study demonstrates that tachyphalaxis does occur to intracerebroventricularly (ICV) applied angiotensins. This change in angiotensin sensitivity may be a result of receptor down regulation and internalization which appear to occur at least in the in vitro preparation used in this study.

High-performance liquid chromatographic analysis of ‘specifically bound’ label after [125I]angiotensin II binding to rat brain membranes

The combined hypothalamus-thalamus-septum and anteroventral third ventricular region (HTSA) of the rat was examined for [125I]angiotensin II ([125I]AII) binding using two protocols: one that preserved synaptosomal structure and a second that did not. Although maximum binding (Bmax) and dissociation constants (Kd) were similar in both preparations, high-performance liquid chromatographic analyses revealed that [125I]AII made up the majority of specifically bound label in the synaptosome preserved preparation while [125I]tyrosine ([125I]Tyr) represented most of the specifically bound label in the disrupted preparation. These results indicate that [125I]Tyr accumulation occurred subsequent to binding and degradation of [125I]AII and are consistent with the notion that rapid internalization of the receptor-[125I]AII complex occurs in those preparations where the synaptosomal structure remains intact.

Central Metabolism of Angiotensins: Potential Functional Significance

Many species2/7 have both pressor and dipsogenic responses to intracerebroventricularly (ICV) applied angiotensin II (AII) and III (AIII). Implicit in this central nervous system (CNS) responsiveness to AII and AIII is the existence of specific membrane-bound receptors. Furthermore these receptors would be expected to be present in those areas of the CNS which are known to be angiotensin sensitive, namely the circumventricular organs4. Surprisingly, this logical inference with regard to the distribution of receptors as determined by radioligand binding methods has not been consistently validated when 125I-AII is used as the labeled ligand in the presence of chelating agents1/3/6. However, when 125I-AIII is used as the radioligand, “apparent” binding is seen in many brain regions in every species examined. The term “apparent” is used because high performance liquid Chromatographic (HPLC) analysis of bound label derived from 125I-AIII incubated membranes clearly indicated that degradation products, especially 125I-tyrosine (Tyr), make up the large majority of bound label. This finding, may, in fact, indicate that “apparent” AIII binding is artifactual in the sense that it has nothing to do with angiotensin binding sites but may represent nonspecific degradation of angiotensins and subsequent uptake of labeled products. On the other hand, this degradation and tyrosine transloction may be a concerted event that accompanies angiotensin binding, suggesting that specific peptidases may be a part of the receptor complex. This study which examines these possibilities strongly supports this notion.