Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ_1–40 carrying the "Dutch" mutation (HCHWA-D Aβ_1–40) as well as wild-type Aβ_1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild-type Aβ_1–40 and HCHWA-D Aβ_1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ-induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA-D Aβ_1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short-term treatment with HCHWA-D Aβ_1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid-binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA-D.     

The relationship between tumor necrosis factor and human immunodeficiency virus gene expression in lymphoid tissue.

In tissue culture models of chronic human immunodeficiency virus type 1 (HIV-1) infection, cytokines such as tumor necrosis factor alpha (TNF-alpha) activate viral gene expression. We sought evidence that TNF-alpha might similarly regulate viral gene expression in vivo in the major lymphoid tissue (LT) reservoir. We used in situ hybridization, quantitative image analysis, and double-label techniques to compare cytokine and HIV-1 RNA levels in sections of tonsil and lymph node tissues obtained from individuals in early and later stages of HIV-1 infection. The levels of TNF-alpha gene expression in LT from HIV-1-infected an uninfected individuals were indistinguishable, and we found no correlation between TNF-alpha gene expression in LT and the level of HIV-1 gene expression in LT. There is thus little evidence that in vivo TNF-alpha significantly influences HIV production in LT.     

Assessing total body and extracellular water from bioelectrical response spectroscopy

Siconolfi, Steven F., Randal J. Gretebeck, William W. Wong,Robert A. Pietrzyk, and Sheril S. Suire. Assessing total body andextracellular water from bioelectrical response spectroscopy. J. Appl. Physiol. 82(2): 704-710, 1997.We developed and validated assessments for total body water(TBW) and extracellular water (ECW) by using two resistance values of anew electric circuit model (CM) (two resistors: a capacitor and aninductor) with or without body mass. Fluid shifts occurring after 40 min of supine rest did not decrease the validity of either estimate. CMestimates were valid; r = 0.941 to0.969, low SE of estimates of 1.15-2.28 kg, nonsignificant meandifferences (CM  dilution; % = 0.4 to 1.3%) thatwere close to the expected measurement errors for TBW (±1%) andECW (±5%), and Bland-Altman pairwise comparisons that showedequivalence between methods. The CM estimates of TBW and ECW hadmarginally better validity than the previously published bioimpedancemodels. The advantage of the CM model is its assessments of multiplefluid spaces and that it does not require gender-specific equations. Weconclude that CM estimate of TBW is acceptable, whereas furthervalidation is needed before the ECW estimate should be used in aclinical or research setting.

     

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo

Reiser, Peter J., William O. Kline, and Pal L. Vaghy.Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82(4): 1250-1255, 1997.Fast-twitch skeletal muscles contain more neuronal-type nitricoxide synthase (nNOS) than slow-twitch muscles because nNOS is presentonly in fast (type II) muscle fibers. Chronic in vivo electricalstimulation of tibialis anterior and extensor digitorum longus musclesof rabbits was used as a method of inducing fast-to-slow fiber typetransformation. We have studied whether an increase in musclecontractile activity induced by electrical stimulation alters nNOSexpression, and if so, whether the nNOS expression decreases to thelevels present in slow muscles. Changes in the expression of myosinheavy chain isoforms and maximum velocity of shortening of skinnedfibers indicated characteristic fast-to-slow fiber type transformationafter 3 wk of stimulation. At the same time, activity of NOS doubled inthe stimulated muscles, and this correlated with an increase in theexpression of nNOS shown by immunoblot analysis. These data suggestthat nNOS expression in skeletal muscle is regulated by muscle activityand that this regulation does not necessarily follow the fast-twitchand slow-twitch pattern during the dynamic phase of phenotypetransformation.

     

Interaction of cross-sectional area, driving pressure, and airflow of passive velopharynx

Isono, Shiroh, Thom R. Feroah, Eric A. Hajduk, Rollin Brant,William A. Whitelaw, and John E. Remmers. Interaction ofcross-sectional area, driving pressure, and airflow of passive velopharynx. J. Appl. Physiol. 83(3):851-859, 1997.Previous studies have shown that, when thepharyngeal muscles are relaxed, the velopharynx is a highly compliantsegment of the pharynx. Thus, under these circumstances,cross-sectional area of the velopharynx (A_VP), drivingpressure across the velopharynx (P), and inspiratory airflow(I) willbe mutually interdependent variables. The purpose of the presentinvestigation was to describe the interrelation among these threevariables during inspiration. We studied 15 sleeping patients withobstructive sleep apnea/hypopnea when the pharyngeal muscles wererendered hypotonic by applying continuous positive airway pressure tothe nasal airway.A_VP, determined by endoscopic imaging, was significantly greater at onset ofI limitationthan at minimum oropharyngeal pressure(P < 0.01). Snoring was neverobserved duringIlimitation. In a subgroup of six patients, values for P,I, andA_VP were obtainedat 0.1-s intervals at various levels of mask pressure. For these sixpatients, the mathematical expressionI = 0.657(A_VP/A_max) · P^0.332,where A_max ismaximal A_VP,described the relationship among the three variables(R² = 0.962) forflow-limited and non-flow-limited inspirations. The impedance of thepassive velopharynx, defined asP^0.33/,was inversely related toA_VP and increaseddramatically when A_VP was <0.3cm². In summary, we observed aprogressive decrease inA_VP during flow-limited inspiration in patients with obstructive sleep apnea. Thisconstriction of the velopharynx contributes to an increase invelopharyngeal impedance that, in turn, counterbalances the increase inP during flow limitation.

     

Nitric oxide and beta -adrenergic agonist-induced bronchial arterial vasodilation

Charan, Nirmal B., Shane R. Johnson, S. Lakshminarayan,William H. Thompson, and Paula Carvalho. Nitric oxide and-adrenergic agonist-induced bronchial arterial vasodilation.J. Appl. Physiol. 82(2): 686-692, 1997.In anesthetized sheep, we measured bronchial blood flow(br) by an ultrasonic flow probe to investigate the interaction between inhaled nitric oxide (NO; 100 parts/million) givenfor 5 min and 5 ml of aerosolized isoetharine (1.49 × 10² M concentration).NO and isoetharine increased br from 26.5 ± 6.5 to 39.1 (SE) ± 10.6 and 39.7 ± 10.7 ml/min,respectively (n = 5).Administration of NO immediately after isoetharine further increasedbr to 57.3 ± 15.1 ml/min. NO synthase inhibitorN-nitro-L-arginine methyl esterhydrochloride (L-NAME; 30 mg/kg, in 20 ml salinegiven iv) decreased br to 14.6 ± 2.6 ml/min. NO given three times alternately with isoetharine progressively increased br from 14.6 ± 2.6 to 74.3 ± 17.0 ml/min, suggesting that NO and isoetharine potentiatevasodilator effects of each other. In three other sheep, afterL-NAME, three sequential doses of isoetharine increased br from 10.2 ± 3.4 to11.5 ± 5.7, 11.7 ± 4.7, and 13.3 ± 5.7 ml/min,respectively, indicating that effects of isoetharine are predominantlymediated through synthesis of NO. When this was followed by threesequential administrations of NO, br increased by146, 172, and 185%, respectively. Thus in the bronchial circulationthere seems to be a close interaction between adenosine3,5-cyclic monophosphate- and guanosine3,5-cyclic monophosphate-mediated vasodilatation.

     

A Genetic Polymorphism in Coumarin 7-Hydroxylation: Sequence of the Human CYP2A Gnes and Identification of Variant CYP2A6 Alleles

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes—CYP2A6, CYP2A7, and CYP2A13—plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6ν2. Sequence differences in the CYP2A6ν2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6ν2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6ν2 allele, and a variant—designated CYP2A6ν1—that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6ν1 and CYP2A6ν2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.