Kinetics of In-111-platelets in the baboon: I. Isolation and labelling of a viable and representative platelet population

A fully representative and viable platelet population was isolated from the blood of 15 baboons by a multiwash procedure, and labelled with In-111-oxine. The recovery of the total platelet population in the circulation was 85% +/- 9. Mean platelet life span was 146 hr +/- 13. Correcting for plasma radioactivity (always less than 3.5%) did not significantly affect the estimate of platelet life span (145 hr +/- 16) or recovery (85% +/- 12). Platelet survival estimates, repeated at different times, were reproducible. In 5 baboons, platelets were also harvested by a single step differential centrifugation. The mean life span of a representative platelet population was significantly longer than that of platelets harvested by a single step. Recovery values of the representative and non-representative population were similar. We conclude that it may be important to harvest and label a fully representative platelet population for kinetic studies. The proposed method is simple and reproducible, and may be applied in studies in humans.     

Angiotensin II binding sites in the hamster brain: localization and subtype distribution

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [¹²⁵I][sar¹, ile⁸]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AAT, subtype) and PD123177 (AT₂ subtype). Binding was quantified by densoritometric autoradiograms and localized by comparison with adjacent thionin stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [¹²⁵I][sar¹, ile⁸]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT₁ binding sites in the hamster but AT₂ in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.     

Validation of the dual radiotracer method for quantitative In-111 platelet scintigraphy

We have developed a simple in vivo scintigraphic technique that permits accurate quantitative comparison of intravascular platelet deposition in blood vessels of similar size. Regional count information from scintigraphic images of In-111 platelets and Tc-99m red blood cells is used to determine the ratio of radioactivity in the thrombus relative to that in the blood. Multiplication of this ratio by the percent injected dose (%ID) of In-111 per ml of blood yields a quantity (%ID index) that is directly proportional to the %ID of In-111 platelets in the thrombus as measured in vitro (r = .879, p less than 0.005). This technique is well suited for quantitative studies of scintigraphically detectable platelet deposition in both animals and in man.     

Kinetics and in vivo distribution of in-111-labelled platelets and platelet function in familial hypercholesterolaemia

The kinetics, in vivo distribution and sites of sequestration of autologous In-111-labelled platelets and other platelet function parameters were studied in ten patients with type IIa or IIb familial hypercholesterolaemia and thrombotic complications of atherosclerosis. The in vitro platelet aggregation response to ADP (P = 0.50) and collagen (P = 0.46); binding of fibrinogen to platelets (P = 0.61); and plasma beta-thromboglobulin levels (P = 0.42) of the patients and normal reference subjects did not differ significantly. The in vivo distribution of In-111-labelled platelets at equilibrium was within normal limits, and at the end of platelet life-span the sequestration pattern of labelled platelets in the reticuloendothelial system was also normal (spleen P = 0.31; liver P = 0.54). There was minimal evidence of in vivo platelet activation: only mean platelet lifespan (MPLS), 195 +/- 57 hours (difference between mean MPLS of patients and controls was 25 hours, with a 95% confidence interval from 23 to 31 hours; P = 0.02); mean platelet platelet turnover, 2298 +/- 824 platelets/microliter/hour (P = 0.005); plasma platelet factor 4 (P = 0.02); and the mean circulating platelet aggregate ratio, 0.8 +/- 0.1 (P = 0.02); differed significantly from normal. These results suggest that abnormalities of platelet function and kinetics observed in type II hyperlipoproteinaemia cannot be ascribed wholly to the hyperlipidaemia, but may be induced by the associated atherosclerosis.     

The distribution of angiotensin II binding sites in rodent brain

The distribution of specific angiotensin II (AII) binding capacity of several brain regions, pituitary, and adrenals was determined in 6 rodent species namely rats, mice, hamsters, kangaroo rats, gerbils and degus. Rats and mice had similar distributions with the highest levels of binding observed in the area postrema, septum and superior colliculi. Low levels were seen in the cortex, cerebellum, striatum and hippocampus. Other areas had intermediate levels. The distribution of AII binding in gerbils and degus was strikingly different from rats and mice. In these species, little or no binding could be detected in the brain. Additionally, the level of binding in degu adrenals was extremely low when compared to the binding observed in the adrenals of the other species. The distribution of AII binding sites in hamsters and kangaroo rats, although similar in some ways to rats and mice, had several major differences. Both had much higher levels of specific binding in cerebellum, striatum, and the hippocampus areas which had low levels of AII binding in rats an mice. Hamsters were the only species to exhibit significant specific binding in the cortex. The kangaroo rats had an unusual distribution of receptors with an apparent lack of specific binding in midbrain and area postrema.     

Comparative PET studies of the kinetics and distribution of cocaine and cocaethylene in baboon brain.

Recent studies have suggested that cocaethylene, an active metabolite of cocaine found in blood and postmortem brain of individuals self-administering cocaine and alcohol, may play a role in the increased toxicity seen when coadministering these 2 drugs. We have used positron emission tomography (PET) and carbon-11 (t1/2:20.4 min) labeled cocaine and cocaethylene to compare the short-term kinetics of cocaine and cocaethylene in baboon brain. The regional uptake of [11C]cocaine cocaethylene in baboon brain. The regional uptake of [11C]cocaine ([11C]COC) and [11C]cocaethylene ([11C]CE), 5-8 mCi and 4-6 micrograms, in baboon brain (n = 7) were similar but clearance from whole brain (global, GL) and from striatum (SR), thalamus (TH), and cerebellum (CB) was slower for cocaethylene. Steady-state distribution volumes (DV) were not significantly different in the striatum but were greater for cocaethylene in the thalamus, cerebellum, and whole brain. Debenzoylation of cocaethylene proceeded at about one-third the rate of cocaine, as determined by in vitro incubation of labeled cocaethylene and labeled cocaine with baboon plasma and with purified horse butyryl-cholinesterase (EC 3.1.1.8). Even though the slower clearance of cocaethylene could lead to longer tissue exposures and potentially accentuated or different physiological effects relative to cocaine, the difference between the 2 drugs is not large. Thus it is more likely that the direct actions of cocaine and alcohol on some organs, rather than cocaethylene, account for this enhanced toxicity.     

Mapping cocaine binding sites in human and baboon brain in vivo

The first direct measurements of cocaine binding in the brain of normal human volunteers and baboons have been made by using positron emission tomography (PET) and tracer doses of [N-11C-methyl]-(-)-cocaine ([11C]cocaine). Cocaine's binding and release from brain are rapid with the highest regional uptake of carbon-11 occurring in the corpus striatum at 4-10 minutes after intravenous injection of labeled cocaine. This was followed by a clearance to half the peak value at about 25 minutes with the overall time course paralleling the previously documented time course of the euphoria experienced after intravenous cocaine administration. Blockade of the dopamine reuptake sites with nomifensine reduced the striatal but not the cerebellar uptake of [11C]cocaine in baboons indicating that cocaine binding is associated with the dopamine reuptake site in the corpus striatum. A comparison of labeled metabolites of cocaine in human and baboon plasma showed that while cocaine is rapidly metabolized in both species, the profile of labeled metabolites is different, with baboon plasma containing significant amounts of labeled carbon dioxide, and human plasma containing no significant labeled carbon dioxide. These studies demonstrate the feasibility of using [11C]cocaine and PET to map binding sites for cocaine in human brain, to monitor its kinetics, and to characterize its binding mechanism by using appropriate pharmacological challenges.     

Distribution of cocaine recognition sites in monkey brain: I. In vitro autoradiography with [3H]CFT.

The cocaine analog [3H]CFT ([3H]WIN 35,428) was used to map and characterize cocaine recognition sites in the squirrel monkey brain by quantitative autoradiography. Coronal tissue sections were incubated with 5 nM [3H]CFT to measure total binding or with [3H]CFT in the presence of 30 microM (-)-cocaine to measure nonspecific binding. High densities of [3H]CFT binding sites were present in dopamine-rich brain regions, including the caudate nucleus, putamen, nucleus accumbens, and olfactory tubercle. In each of these regions specific binding was greater than 90% of total binding. Several additional brain regions exhibited intermediate densities of [3H]CFT binding, including the substantia nigra, the zona incerta, the amygdala, and the hypothalamus. Low, though measurable levels of binding were observed in the bed nucleus of the stria terminalis, the ventral tegmental area, the medial preoptic area, the pineal, the hippocampus, and thalamic central nuclei. Near-background levels of binding were found in white matter, cortical regions, globus pallidus, and cerebellum. The pharmacological specificity of [3H]CFT binding in various brain regions was determined in competition studies using [3H]CFT and a range of concentrations of selected monoamine uptake inhibitors. In all brain regions examined, stereoselective inhibition of [3H]CFT binding was observed for the (-) over the (+) isomer of cocaine. For other drugs tested, competition experiments indicated a rank order of potency of GBR 12909 greater than or equal to CFT greater than bupropion, suggestive of binding of [3H]CFT to elements of the dopamine transport system. The results demonstrate that although densities of [3H]CFT binding sites are highest in the caudate nucleus, putamen, and nucleus accumbens/olfactory tubercle, significant levels of binding can be detected in other brain regions that may contribute to the behavioral and physiological effects of cocaine.     

Progressive supranuclear palsy and In111-DTPA cisternography.

Three cases of progressive supranuclear palsy are reported in which In111-DTPA cisternography showed slow diffusion, ventricular reflux and failure of isotope clearance. The clinical diagnosis of progressive supranuclear palsy was confirmed histologically in two of these patients. The possible causes of the cisternographic changes and their relationship to the changes of CSF dynamics in progressive supranuclear palsy are discussed.     

In vitro autoradiographic localization of calcitonin binding sites in human medulla oblongata

In this study we examined the distribution of calcitonin (CT) binding sites in the human medulla oblongata by in vitro autoradiography. In competition studies, the rank order of potency for calcitonins competing for ¹²⁵I-salmon CT binding was salmon CT > porcine CT > human CT, which is consistent with physiologically relevant CT receptors in other systems. For the determination of CT binding in the human medulla, 20-μm cryostat sections were incubated with ¹²⁵I-salmon CT in the presence or absence of 10^?6 M unlabelled salmon CT to map specific CT binding sites. Punctate binding was observed over discrete nuclei of the medulla. High binding densities were seen over subnuclei of the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the intermediate reticular zone, the gigantocellular and dorsal paragigantocellular nuclei, and the raphe obscurus nucleus. Moderate levels of binding were observed over the lateral paragigantocellular nucleus and the rostral extent of the epiolivary nucleus. The cuneate and gracile nuclei and the fiber tracts did not contain detectable binding, while the inferior olivary nucleus had moderate levels of nonspecific binding. The localization of calcitonin binding sites in the human presents similarities but also important differences to the distribution in rat and cat. The most notable difference is the extreme binding densities in the intermediate reticular zone of the human. The location of binding sites suggests involvement of calcitonin in regulation of autonomic function.     

Red blood cell choline. II: Kinetics in Alzheimer's disease

The kinetic parameters of choline uptake into red blood cells from patients with Alzheimer's disease and normal elderly controls were compared. The Kd and Vmax values for choline uptake into red cells were determined based on a kinetic analysis of choline uptake at six different concentrations of labeled extracellular choline. The theoretical choline uptake, representing the initial rate of choline influx into choline-depleted red cells given the plasma choline concentration and the kinetic parameters of choline uptake, was also calculated. Alzheimer's disease patients and normal controls did not differ in any kinetic parameter of choline uptake. Kd and Vmax values for red cell choline uptake were strongly correlated among normal controls, but not among patients with Alzheimer's disease. In addition, among the patients with Alzheimer's disease, the theoretical choline uptake was strongly correlated with the severity of dementia. The possible significance of these findings in relation to altered choline metabolism in Alzheimer's disease is discussed.     

Characterization and distribution of angiotensin II binding sites in fetal and neonatal astrocytes from different rat brain regions.

Although angiotensin II (Ang II) binding sites have been extensively investigated in brain, revealing the presence of both AT1 and AT2 subtypes in various areas, the question as to which cells express AT1 and AT2 sites is still open. We report here that primary cultures of astrocytes obtained from various brain regions of fetal (F17) and one-day-old rats express Ang II binding sites belonging only to the AT1 subtype. The binding sites have the same binding profile in all regions tested; however, much less binding was observed in membranes of astrocytes derived from cortical than from subcortical regions and almost none were found in neonatal cortex. In addition, the dispersion method used at the onset of culture affects the number of binding sites present at the end of the culture period.     

Autoradiographic distribution of TRH binding sites in the human hypothalamus.

Using in vitro quantitative autoradiography and [3H]3MeTRH, a selective high affinity radioligand, we examined the rostrocaudal distribution of TRH binding sites in both the infant and the adult human hypothalamus. The saturation curve shows that the [3H]3MeTRH binds with high affinity to a single class of TRH binding sites and is saturable, the apparent constant of dissociation is in the namomolar range. TRH binding sites showed a wide distribution, principally in the anterior and mediobasal levels of the hypothalamus. TRH binding site concentration was highest within the diagonal band of Broca, the lateral preoptic area, the infundibular and the tuberal nuclei. TRH binding site concentration was moderate in the ventromedial nucleus and the medial preoptic area, whereas we observed low densities in the periventricular, paraventricular and mammillary nuclei. The distribution in the infant and the adult is generally similar. However, it is noteworthy that the infant tuberal nuclei displayed a lower binding site density when compared to the adult. On the other hand, the diagonal band of Broca is relatively more labeled in infant. The analysis of the whole hypothalamus allows us to ascertain the absence of lateral asymmetric distribution both in the infant and the adult. No significant difference is noticed when considering as parameters of variation age, sex or post mortem delay.     

Influence of anatomic origin on intracranial distribution of micro-emboli in the baboon

The purpose of this study was to investigate whether the anatomic origin of micro-emboli influences their intracranial distribution. In twenty-two baboons, we examined the distribution of 99-Technetium labelled albumin aggregates (5 to 40 microns in size) after injection into the circulation at the left atrium (LA), carotid trifurcation (CA), and anterior and posterior common carotid artery (CCI). In a further subgroup, the emboli were introduced at the carotid trifurcation with the contralateral carotid artery ligated (CA + L). The results of this study demonstrated that aggregates introduced at the carotid artery lodged preferentially in the ophthalmic (p = 0.032) and middle cerebral artery territories (p = 0.016). If the contralateral common carotid artery was ligated, however, more aggregates were found in the ipsi- and contralateral anterior cerebral artery territories (p = 0.01, p = 0.003). Aggregates introduced into the cardiac circulation were equally distributed throughout the brain. This experimental model determined patterns of flow that might be analogous to the human situation where unilateral or bilateral carotid stenosis or stenosis with contralateral occlusion has occurred or embolus from cardiac source has occurred. The results do not imply that the 40 micron microaggregates do cause TIA. These experimental findings support clinical observations that cardiac lesions may cause transient ischemic attacks (TIA) anywhere in the brain. In contrast, those of carotid artery origin cause predominantly middle cerebral or ophthalmic artery territory TIAs unless the contralateral carotid artery is severely stenosed or occluded.     

Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography.

The distribution of galanin (GAL) binding sites in the visual cortex of cat and monkey was determined by autoradiographic visualization of [125I]-GAL binding to tissue sections. Binding conditions were optimized and, as a result, the binding was saturable and specific. In cat visual cortex, GAL binding sites were concentrated in layers I, IVc, V, and VI. Areas 17, 18, and 19 exhibited a similar distribution pattern. In monkey primary visual cortex, the highest density of GAL binding sites was observed in layers II/III, lower IVc, and upper V. Layers IVA and VI contained moderate numbers of GAL binding sites, while layer I and the remaining parts of layer IV displayed the lowest density. In monkey secondary visual cortex, GAL binding sites were mainly concentrated in layers V-VI. Layer IV exhibited a moderate density, while the supragranular layers contained the lowest proportion of GAL binding sites. In both cat and monkey, we found little difference between regions subserving central and those subserving peripheral vision. Similarities in the distribution of GAL and acetylcholine binding sites are discussed.     

Responses of baboon cerebral and extracerebral arteries to prostacyclin and prostaglandin endoperoxide in vitro and in vivo.

The responses of baboon cerebral and extracerebral arteries to prostaglandin endoperoxide (PGH2) and prostacyclin (PGI2) were investigated on isolated arteries and in vivo by serial angiography. Both PGH2 and PGI2 could produce dose-dependent contraction or relaxation of isolated arteries. PGH2 induced relaxation was indicative of prostacyclin synthetase activity, the enzyme which converts PGH2 to PGI2. In isolated arteries tested one to four hours post mortem only the vertebral artery showed prostacyclin synthetase activity. Thus PGH2 induced contraction of cerebral arteries may be indicative of a physiological function. Vasomotor tone may in part be the result of a balance between PGH2 constriction and PGI2 dilatation. In vivo PGI2 infusion caused pronounced and prolonged dilatation of cerebral arteries, which lasted longer than the cardiovascular changes. As PGI2 is the most potent cerebral vasodilator drug tested, it may be of clinical use in the treatment of cerebral vasospasm.     

Learning and behavioral abnormalities in the seizure-prone baboon.

A high incidence of behavioral and learning difficulties has been found in juvenile seizure-prone baboons presented with a graded series of operant behavioral task challenges. The animals fell into two distinct groups based upon the rate at which their operant performance was able to develop, the amount of day-to-day variability in performance level, their ability to tolerate change, and the frequency of appearance of symptoms of emotionality. The association between learning problems and behavior problems in these seizure-prone baboons may correlate well with the deficits in both intellectual performance and social behavior that have been reported in some studies with human epileptics. In addition, the data suggest a relationship between familial factors and trainability, and between predisposition to seizures and trainability.     

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina.

The distribution of anionic sites on the cell membranes and basal laminae of vascular endothelial cells in the rat sciatic nerve was investigated using cationic ferritin (CF) and cationic colloidal gold (CCG). Nerves fixed by perfusion followed by immersion were chopped into 400 microns thick slices and incubated in CF or embedded in LR White resin for staining with CCG. Using electron microscopy, the distribution of these tracers was investigated. The results indicated that microdomains of various charge densities exist. Diaphragms of caveolae and transendothelial channels, and luminal endothelial processes are highly anionic, the basal laminae of endothelial cells and pericytes and luminal membranes are medium and abluminal membranes least anionic. Inter-endothelial tight junctions were unlabelled and not penetrated by CF. These structures are thought to represent charge and size filters that control permeability of the vasa nervorum. The distribution of these charge-size filters is discussed in terms of the blood-nerve barrier, a physiological property present in the endo- but absent in the peri- and epineurial vessels.