Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules.

Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of approximately 0.05 to 0.15 micrometer Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy. The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values. A similar distribution was obtained after perfusion of H1 or H2 receptor agonists coupled to ferritin (2-pyridylethylamine-ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively). The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H1 and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H2 receptors.

Comparative demonstration of pulmonary fat emboli by "en bloc" osmium tetroxide and oil red O methods.

The presence and localization of fat in human lung tissue was evaluated by the "en bloc" staining procedure with osmium tetroxide performed with formalin fixed tissue with subsequent paraffin sectioning and with the oil red O technique performed with frozen sections. Fifty-one autopsy cases were divided into three clinical groups: group A, consisting of 17 patients with severe skeletal trauma; group B, consisting of 14 patients with minor skeletal trauma; and group C, consisting of 20 control patients without trauma. Adjacent sections of lung were selected from all cases, stained with the "en bloc" osmium tetroxide and oil red O methods, coded, and examined under the light microscope without knowledge of the clinical grouping, Stainable fat was graded on a 1 to 4+ scale, and attention was given to histologic localization in tissue sections. The "en bloc" osmium tetroxide technique revealed greater amounts of stainable lipid in clinical groups A and B and was most effective in demonstrating lipid when present in small quantities (group B). Since the method permits the employment of paraffin sections, evaluation of fine histologic detail is an advantage that is not always obtained in frozen sections. It is concluded that the "en bloc" osmium tetroxide technique is superior to the oil red O technique in terms of visualization and histologic localization of minute amounts of lipid in lung tissue.

Distribution and origins of VIP-immunoreactive nerves in the cephalic circulation of the cat.

VIP-immunoreactive (IR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within microganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons.

Neurochemical studies on the existence, origin and characteristics of the serotonergic innervation of small pial vessels.

Substantial concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), comparable to those found in brain tissue, were measured in the small pial vessels of the rat, rabbit and cat. Both rat and rabbit pial vessels exhibited a high affinity uptake process with kinetic parameters similar to those identified for the cerebral cortex. Labelled 5-HT, taken up by isolated rabbit pial vessels was released, in a calcium-dependent manner, by potassium-induced depolarization. Various pharmacological manipulations were carried out in the rat. Systemic administration of the 5-HT precursor, 5-hydroxytryptophan and the monoamine oxidase inhibitor, pargyline, significantly increased the concentration of 5-HT in the pial vessels; in contrast, two depleting agents (p-chloroamphetamine and reserpine) and the tryptophan hydroxylase inhibitor, p-chlorophenylalanine, all decreased the perivascular 5-HT levels. A serotonergic antagonist (methysergide) and a 5-HT receptor agonist (MK 212) respectively increased and decreased the concentrations of 5-HIAA in the pial vessels. These pharmacologically induced changes observed in pial vessels were not dissimilar from those noted for cortical tissue. Electrolytic lesions of the nuclei raphes medianus and/or dorsalis markedly decreased the levels of 5-HT and 5-HIAA in these small cerebral arterioles. Electrical stimulation of these nuclei decreased 5-HT although 5-HIAA concentrations tended to increase. A number of conclusions may be drawn from these studies. Thus, there is a serotonergic innervation of the cerebral circulation in several laboratory species which unequivocally originates in the raphé nuclei. Furthermore, these perivascular fibres possess synthetic, storage, release, inactivation and autoregulatory processes for 5-HT which, when further elucidated, may offer some rationale for the treatment of those cerebrovascular diseases in which this neurotransmitter and vasoactive agent is believed to be of pathological importance.

Features of the cerebral vascular pattern that predict vulnerability to perfusion or oxygenation deficiency: an anatomic study.

In an ongoing study of brain microvasculature in humans at autopsy, we had the opportunity to analyze the overall scheme of this vascular supply. The native endothelial membrane enzyme, alkaline phosphatase, is used to precipitate black lead sulfide salt in the vessel wall, rendering the brain microvasculature visible by both light microscopy and microradiography. There are six distinct patterns of intraparenchymal afferent blood supply to the supratentorial brain: short arterioles from a single source (e.g., those in the cortex); short- to intermediate-length arterioles, single source (anterior two-thirds of the corpus callosum); short- to intermediate length arterioles and arteries, dual source (subcortical U fibers); intermediate-length arterioles and arteries, triple source (extreme/external capsule and claustrum); long arteries and arterioles, single source (centrum semiovale); and large, long muscular arteries, single source (thalamus and basal ganglia). The nature of this arrangement offers some protection to certain regions of the cerebrum from circulatory challenges such as hypotension, while leaving other areas vulnerable. The distal arterioles supplying two of these protected regions, the U-fiber area and the extreme/external capsule and claustrum area, also exhibit the feature of interdigitation, which can offer additional collateral potential from one arteriolar territory to the next. Aging, hypertension, diabetes mellitus, and atherosclerosis can have a significant impact on brain microcirculation. The way in which vascular patterns dictate the distribution of these effects is discussed. The ability to stain the cerebral microvessels and demonstrate the finer points of their patterns in sections and microradiographs has enabled us to resolve some long-standing questions about vascular connections and directions.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the regional cochlear blood flow in the rat cochlea using non-radioactive microspheres and serially sectioned cochleas.

The regional blood flow to the rat cochlea has been studied using a method which combines the microsphere method with observation of serially-sectioned, plastic-embedded cochleas. Direct quantitation of the microspheres in a reference blood sample and in the different vascular areas of the cochlea allows the analysis of blood flow patterns with respect to the different capillary beds.

Sudden-death ischemic heart disease and dietary magnesium intake: is the target site coronary vascular smooth muscle?

The etiology of sudden-death ischemic heart disease (SDIHD) remains an enigma. Data will be presented which suggest that SDIHD may be due to hypomagnesemia in and around the coronary arterial and arteriolar vessels. We have found that blood vessels (especially arteries and arterioles) deficient with respect to Mg can undergo constriction and spasm; the greater the reduction in Mg2+, the greater the magnitude of the spontaneous contractile responses. The higher the Ca2+:Mg2+ ratio, the greater are the magnitudes of these contractile responses. A severe deficit in surface membrane Mg2+, in particular, results in intense vasospasm. Using direct in situ high resolution microscopy (3000 x), we have found that a lowering of Mg2+ around perfused arterioles (15--20 microns i.d.) will also result in spontaneous vasoconstriction and, in addition, increased arteriolar resistance, tissue ischemia and reduced venous outflow. We have also found that the constrictor actions of certain circulating vasoconstrictor hormones (i.e., angiotensin, serotonin, acetylcholine) are enhanced when [Mg2+] is lowered below the levels normally found in plasma. Other direct studies, from our laboratory, indicate that [Mg2+]o regulates calcium exchange and content of vascular smooth muscle. In summary, the concept to be presented suggests that a deficiency in dietary Mg2+ is a key factor in the high incidence of mortality noted in SDIHD in nations of the Western world. The hypomagnesemia produces progressive vasoconstriction, vasospasm and ischemia, which, given time, will lead to SDIHD.

[Distribution of adrenoreceptors in the microcirculatory components of the circulatory bed of the kidney in the cat]. / Raspredelenie adrenoretseptorov v mikrotsirkuliatornykh zven'iakh krovenosnogo rusla pochki u koshki.

Topography of alpha- and beta-adrenoreceptors and their significance for smooth muscle cells of various links of the arterial division of the cat kidney blood channel were studied by means of alpha- and beta-adreno-stimulators and adrenoblockers administration under the control of general arterial pressure. The initial divisions of the major arteries and arterioles and their primary branches of the kidney fibrous capsule, distal divisions of afferent glomerulus vessels and their corresponding precapillary arterioles as well as the initial divisions of the straight arterioles of the kidney substance medullaris were shown to contain beta-adrenoreceptors. Alpha-adrenoreceptors are present in all other extra-parenchymatous and parenchymatous arteries and arterioles.

Splenic arteriolar hyalin in children.

Sections from 447 children's spleens were examined for arteriolar hyalin. Hyalin is not found in the first year of life, but by the fifth year of life 60 per cent of cases show it. The positive cases include a wide variety of pathological conditions. The development of arteriolar hyalin in the spleen is age dependent, occurs early in life, and is not a pointer to any particular pathological condition.

Immunohistochemical study of cerebral amyloid angiopathy. II. Enhancement of immunostaining using formic acid pretreatment of tissue sections.

Cerebral amyloid angiopathy (CAA) is a biochemically heterogeneous entity most commonly associated with stroke syndromes, Alzheimer's disease (AD), Down's syndrome, and miscellaneous neurologic conditions. The authors have applied and extended (using formic acid pretreatment of histologic sections) an immunocytochemical technique that used antibody to a synthetic 28-amino acid peptide representing a segment of the AD amyloid precursor, to study CAA and related parenchymal amyloid deposits in brain tissues originally derived from: 1) patients with CAA with or without typical clinicopathologic features of AD, cerebral hemorrhage, and infarcts; 2) a young boy with angiocentric brain amyloid; 3) patients with familial (Icelandic, Dutch) forms of cerebral hemorrhage caused by CAA; and 4) Japanese patients with nonfamilial CAA-related brain hemorrhage, sometimes associated with histopathology characteristic of AD. Formic acid pretreatment of sections resulted in markedly enhanced staining of senile plaque core and microvascular, especially capillary, amyloid, and some apparent staining of the neuritic component of senile plaques. Perivascular halos of immunoreactive material were observed frequently. Neurofibrillary tangles were not immunolabeled, nor were blood vessels or any parenchymal components within cerebral white matter. CAA in Japanese patients with nonfamilial encephalic hemorrhages appeared immunocytochemically identical to AD-related CAA. Arterioles in brains that had severe CAA frequently showed significant stenosis of their lumina by nonamyloid hyaline or cellular material.

Vascular glycosaminoglycans in periventricular leukoencephalopathy.

Three cases of adult dementia with periventricular leukoencephalopathy demonstrated staining with alcian blue dye (alcianophilia) in thickened cerebral arteries and arterioles of the abnormal white matter. The property of alcianophilia identifies glycosaminoglycans (GAGs); retention of alcian blue staining at high MgCl2 concentrations (0.7 M) in these cases indicates that the GAGs are highly sulfated and are likely to represent heparan sulfate. Arteriosclerotic risk factors (including hypertension) were absent in all cases, suggesting that GAG deposition occurred as part of a separate cerebral vasculopathic process. These findings suggest that cerebral white matter changes associated with dementia do not always represent Binswanger's disease.

The afferent glomerular arteriole: immunocytochemical and electrophysiological investigations.

Immunocytochemical techniques were used for the localization of the different components of the renin-angiotensin system (RAS) within the kidneys of various species. Special attention was paid to the renin-secreting granulated cells located mainly in the media of the afferent glomerular arteriole. It was demonstrated that variations of kidney renin caused by stimulation or inhibition of the RAS are reflected in changes of the length of the renin-positive part of the afferent arteriole upstream from its entry into the glomerulus. During stimulation, plain smooth-muscle cells are transformed into renin-generating granulated cells. Likewise, the marked species differences in kidney renin are paralleled by corresponding differences in the renin-positive part of the afferent vessel. In this context, the site of action and the relative importance of the stimuli inducing renin secretion, i.e., the beta 1-adrenoreceptor, the mechanoreceptor, and the so-called macula densa mechanism are discussed. Whereas the microtopography of the RAS in the kidney has been at least partly understood, little is known about the stimulus-secretion coupling at the level of the individual granulated cell. Thus, adequate electrophysiological data about granulated cells might help to understand some still obscure phenomena, e.g., the inhibitory effect of Ca2+ on renin secretion. Our investigations in the hydronephrotic kidney preparation of the mouse show that granulated cells do not differ significantly from "plain" smooth-muscle cells in their electrical characteristics. They have a membrane potential of -55 mV and are spontaneously active. Both cell types are depolarized by noradrenaline and angiotensin II (AII), although they remain unaffected by isoproterenol. Since isoproterenol is known to stimulate renin secretion, our results indicate that stimulus-secretion coupling via the beta 1-adrenoreceptor is likely to proceed without changes of membrane potential in granulated cells.

Silver deposition in arteriolar basal laminae in the cerebral cortex of argyric rats.

Electron microscopy of the parietal cortex and subcortical white matter of argyric rat brain revealed extremely fine 10--15 nm deposits of silver within the basal laminae of some arterioles. Stab-wound injury to argyric rats resulted in increased silver deposits in arteriolar basal laminae but not in any deposition of silver in the brain parenchyma.

In situ analysis of alpha-adrenoceptors on arteriolar and venular smooth muscle in rat skeletal muscle microcirculation.

The purpose of this study was to determine whether both alpha 1- and alpha 2-adrenergic receptors exist on vascular smooth muscle of microvessels and whether adrenergic constriction of anatomically distinct microvascular segments is differentially subserved by either receptor subtype. The cremaster skeletal muscle of anesthetized rats was acutely denervated and suspended in a Krebs bath containing cocaine, normetanephrine, and propranolol to block uptake1, uptake2, and beta-receptors, respectively. Intravital microscopy was used to study large distributing arterioles (mean diameter, 100 microns), small precapillary arterioles (25 microns), and capacitance venules (140 microns). Concentration-response (diameter change) curves were obtained for bath-added agonists norepinephrine (mixed alpha 1/alpha 2), phenylephrine (alpha 1), and B-HT 933 (alpha 2) in the absence or presence of antagonists prazosin (alpha 1) and yohimbine (alpha 2). Apparent pD2(-log ED50) values for large arterioles and venules were, respectively, as follows: norepinephrine (7.41 and 7.15), phenylephrine (5.95 and 5.41), and B-HT 933 (5.05 and 5.06). Low concentrations of prazosin (10(-8) M) and yohimbine (10(-7) M) produced receptor subtype-selective antagonism and parallel, dextral displacement of norepinephrine curves for large arterioles and venules. The large arteriole pKB (-log KB) was 7.83 +/- 0.65 for prazosin and 7.36 +/- 0.46 for yohimbine. Higher concentrations of prazosin (10(-7) and 3 X 10(-7) M) and yohimbine (10(-6) M) produced further dextral but nonparallel displacement of norepinephrine curves. In contrast, receptor subtype-selective concentrations of only yohimbine inhibited adrenergic constriction of small, precapillary arterioles; but prazosin had no effect at receptor subtype-selective concentrations. These data suggest that adrenergic regulation of large arterioles and venules in skeletal muscle uses both alpha 1- and alpha 2-adrenoceptors. Precapillary arterioles, however, may be subserved predominantly by alpha 2-receptors.

Vasoactive material present in the glomerula caudalia of the Vervet monkey.

Complex encapsulated neurovascular structures, glomus organs, occur in superficial and deep positions in many species. No direct evidence for their function exists. In skin they may serve thermoregulation by the reason for their structural complexity remains obscure. The concept of a local humoral circulatory control mechanism postulated on the basis of a very high acetylcholine content reported for human glomera coccygia has been investigated in homologous caudal glomus organs in monkey. No acetylcholine was detected. The organs contained 3-4 times the concentration of catecholamine (mainly noradrenaline) found in nearby vessels. Neither structure nor content of vasoactive component gives any support for an endocrine function.

Immunofluorescent localization of type-V collagen as a fibrillar component of the interstitial connective tissue of human oral mucosa, artery and liver.

Polyclonal antibodies against native human type-V collagen were produced in rabbits and goats. Following purification, crossreaction of the antibodies with highly immunogenic peptides of basement membranes or the interstitial matrix was excluded on the basis of sensitive radioimmunoassays. These antibodies, when applied to cryostat sections of human oral mucosa, liver and arterial walls, never stained basement membranes as did antibodies against type-IV collagen or laminin. On the contrary, we observed delicate arborizing fibers in the interstitial compartment with extensions contacting structures such as subepidermal basement membranes. Arterioles contained a unilamellar sheath of longitudinally oriented fibers limited to the intimal layer. Larger arteries exhibited a multilamellar fibrous fluorescence over the whole intima, whereas the media showed a much weaker staining. The data identified type-V collagen as an interstitial fibrillar collagen rather than a basement membrane collagen, with a tissue pattern completely different from that of collagens types I, III, VI or fibronectin. A reinterpretation of the role of type-V collagen in connective tissue function is warranted.

Renin status of the afferent arteriole and ultrastructure of the juxtaglomerular apparatus in 'superficial' juxtamedullary nephrons from rats.

The present light (LM) and transmission electron microscopic (TEM) studies were carried out to further document the anatomy of the 'superficial' juxtamedullary nephrons (SJMNs) located on the inside cortical surface of the rat kidney. TEM revealed that SJMNs possess all vascular and tubular cell types constituting the juxtaglomerular apparatus (JGA) in typical cortical and juxtamedullary nephrons (JMNs). Proximal to the glomeruli, epithelioid cells filled with secretory granules predominated in the media of afferent arterioles. The presence of renin in the granules was immunocytochemically demonstrated by the protein A-gold method. Further upstream from the glomeruli, epithelioid cells alternated with plain smooth muscle cells. The use of renin and angiotensin II antisera revealed similar arteriolar distributions of renin and angiotensin II positive cells in SJMNs as well as in typical JMNs. Numerous nerve terminals were found along afferent and efferent arterioles, suggesting a dense innervation of these vessels. The distribution of renin-containing (i.e., epithelioid) cells in the preglomerular arterioles was assessed in various nephron populations by LM using the PAP method and renin antiserum. In SJMNs, most renin-positive cells were found in the vicinity of the JGA along a mean arteriolar length of 35 +/- 3 micron (range 7-107 micron). In JMNs, renin-positive cells had a similar distribution along a mean arteriolar length of 35 +/- 1 micron. Scattered renin-positive cells were observed up to a maximal arteriolar length of 173 and 238 micron in SJMNs and JMNs, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid angiopathy of Alzheimer's disease: amino acid composition and partial sequence of a 4,200-dalton peptide isolated from cortical microvessels.

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-2 adrenergic receptor localization in the rat heart and kidney using autoradiography and tritiated rauwolscine.

To study the distribution and characterization of alpha-2 adrenergic receptors in the rat heart and kidney, we used light microscopic autoradiography and a computer-based image analyzer to quantify the localization of [3H]rauwolscine (RAUW) binding. Scintillation spectrometry of frozen sections of rat kidney demonstrated rapid binding, saturability, stereospecificity and agonist and antagonist binding characteristic of an alpha-2 adrenergic receptor. For autoradiography, sections of rat kidney and heart were incubated in several concentrations of [3H]RAUW in the absence of (total binding) and in the presence of (nonspecific binding) 10(-5) M yohimbine. The sections were processed and grain density quantified using a computer-based image analyzer. The tubules in the renal cortex had significantly more specific [3H]RAUW labeling than either the renal glomeruli or the tubules in the renal medulla at all concentrations of [3H]RAUW used (P less than .0001). Nonspecific binding was significantly higher over the cortical tubules than either the glomeruli or the tubules in the renal medulla (P less than .0001). Scatchard analysis of specific grain densities determined that the tubules in the renal cortex had the highest density of any structure studied [maximum binding (Bmax) = 1182 grains/10(-2) mm2]. The glomeruli had a Bmax of 485 grains/10(-2) mm2, whereas the tubules in the renal medulla had a Bmax of 273 grains/10(-2) mm2. There were no significant differences among these three regions in the dissociation constant of the [3H]RAUW. When analyzing the heart, we found no specific [3H]RAUW labeling over either the cardiac myocytes or the myocardial arterioles.(ABSTRACT TRUNCATED AT 250 WORDS)