Proteomic analysis of nuclei dissected from fixed rat brain tissue using expression microdissection.

Expression microdissection (xMD) is a high-throughput, operator-independent technology that enables the procurement of specific cell populations from tissue specimens. In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate (EVA) film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p < 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.

Interpretation of pediatric lung function: impact of ethnicity.

RATIONALE: To evaluate the appropriateness of spirometric and plethysmographic reference equations in healthy young children according to ethnic origin. METHODS: Spirometry data were collated in 400 healthy children (214 Black and 186 White) aged 6-12 years. Of these children, 68 Black and 115 White children also undertook plethysmography. Results were expressed as percent predicted according to commonly used equations for spirometry and plethysmography. RESULTS: Black children had lower lung function for a given height compared to White children. The magnitude and direction of these differences varied according to specific outcome. In the studied age range (6-12 years) the ethnic-specific Wang equations were adequate for spirometry (mean results approximating 100% predicted in both ethnic groups). By contrast, significant differences were found between observed and % predicted plethysmographic lung volumes according to published equations derived from White children: Among the Black children, function residual capacity (FRC) and total lung capacity (TLC) were on average, 14 and 6% lower than predicted, whereas mean residual volume (RV) and RV/TLC were 4 and 10% higher. Among White children, the Rosenthal equations gave the best fit, with the exception of FRC which was, on average, 9% lower than predicted. CONCLUSION: Spirometry equations may suffice in Black children; however, interpretation of static lung volumes in Black children is limited due to inappropriate reference equations. More appropriate plethysmographic reference equations that are applicable to all ethnic groups across the entire age range are urgently needed.

Occupational UVR skin protection by make-up.

New legislation has been introduced in the Member States of the European Union, covering worker exposure to artificial optical radiation. Use of make-up could reduce the ultraviolet hazard level and provide additional protection for skin against UV radiation (UVR). This is particularly important in entertainment and filming where intentional exposure of the actors and presenters to the very intense light sources may be required for extended periods of time. This document presents the assessment of UVR protection of make-up used in entertainment and demonstrates that the protection level varies considerably for different luminaires and application techniques. An important practical implication of this finding is that make-up alone cannot be considered as a reliable protection measure against skin exposure to actinic UV.

Assessment of personal exposures to optical radiation in large entertainment venues.

Workplace exposure to optical radiation from artificial sources is regulated in Europe under the Artificial Optical Radiation Directive 2006/25/EC implemented in the UK as The Control of Artificial Optical Radiation at Work Regulations 2010. The entertainment environment often presents an extremely complex situation for the assessment of occupational exposures. Multiple illumination sources, continuously changing illumination conditions and people moving during performances add further complexity to the assessment. This document proposes a methodology for assessing the risks arising from exposure to optical radiation and presents detailed case studies of practical assessment for two large entertainment venues.

Utility of multivariate analysis in support of in vitro metabolite identification studies: retrospective analysis using the antidepressant drug nefazodone.

The utility of multivariate analysis in in vitro metabolite identification studies was examined with nefazodone, an antidepressant drug with a well-established metabolic profile. The chromatographic conditions were purposefully chosen to reflect those utilized in high-throughput screening for microsomal stability of new chemical entities. Molecular ion, retention time information on groups of human liver microsomal samples with/without nefazodone was evaluated by principal component analysis (PCA). Resultant scores and loadings plots from the PCA revealed the segregation and the ions of interest that designated the drug and its corresponding metabolites. Subsequent acquisition of tandem mass spectrometry (MS/MS) spectra for targeted ions permitted the interrogation and interpretation of spectra to identify nefazodone and its metabolites. A comparison of nefazodone metabolites identified by PCA versus those found by traditional metabolite identification approaches resulted in very good correlation when utilizing similar analytical methods. Fifteen metabolites of nefazodone were identified in beta-nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented human liver microsomal incubations, representing nearly all primary metabolites previously reported. Of the 15 metabolites, eight were derived from the N-dealkylation and N-dephenylation of the N-substituted 3-chlorophenylpiperazine motif in nefazodone, six were derived from mono- and bis-hydroxylation, and one was derived from the Baeyer Villiger oxidation of the ethyltriazolone moiety in nefazodone.

[Eye fundus autofluorescence technique in the early diagnosis of age-related macular degeneration].

Autofluorescence (AF) of the eye fundus is one of the most promising studies. AF provides specific molecular information on the retinal pigment epithelium and enables one to diagnose early phenotypic changes that are predictors for progression of age-related macular degeneration. Many investigations have demonstrated that AF is a valuable clinical technique that should be improved in order to have information accessible to a patient on the diagnosis and prediction of age-related macular degeneration.

Eradication of Helicobacter spp. from a rat breeding colony.

Although Helicobacter spp. have been viewed as bacteria with low pathogenicity, many investigators have shown that these low-grade pathogens have the potential to become a severe threat in immunocompromised, inbred, and transgenic animals. Therefore the presence of Helicobacter spp. in experimental animals is considered to be an unacceptable variable. In this study a formulation of medicated feed was designed and tested in an attempt to eradicate Helicobacter spp. from an infected rat breeding colony. Two feeding protocols were used: 1) treating Helicobacter-infected pregnant dams to produce clean offspring and 2) treating infected adult animals long enough to eliminate the organisms. Bacterial DNA was extracted from feces and amplified using primers that recognized the Helicobacter spp.-specific region of the 16S rRNA gene. Fecal samples from the weanlings from protocol 1 tested negative for Helicobacter spp. at 1 week before and 2 and 12 weeks after weaning. Infected adult rats from protocol 2 tested negative after three cycles of 2 weeks on and 2 weeks off the medicated feed. Animals from both protocols have remained Helicobacter-free for 8 months.

General statistics of stochastic process of gene expression in eukaryotic cells.

Thousands of genes are expressed at such very low levels (< or =1 copy per cell) that global gene expression analysis of rarer transcripts remains problematic. Ambiguity in identification of rarer transcripts creates considerable uncertainty in fundamental questions such as the total number of genes expressed in an organism and the biological significance of rarer transcripts. Knowing the distribution of the true number of genes expressed at each level and the corresponding gene expression level probability function (GELPF) could help resolve these uncertainties. We found that all observed large-scale gene expression data sets in yeast, mouse, and human cells follow a Pareto-like distribution model skewed by many low-abundance transcripts. A novel stochastic model of the gene expression process predicts the universality of the GELPF both across different cell types within a multicellular organism and across different organisms. This model allows us to predict the frequency distribution of all gene expression levels within a single cell and to estimate the number of expressed genes in a single cell and in a population of cells. A random "basal" transcription mechanism for protein-coding genes in all or almost all eukaryotic cell types is predicted. This fundamental mechanism might enhance the expression of rarely expressed genes and, thus, provide a basic level of phenotypic diversity, adaptability, and random monoallelic expression in cell populations.

Biomarker risk assessment and bladder cancer detection in a cohort exposed to benzidine.

BACKGROUND: Cancer screening with highly sensitive, specific biomarkers that reflect molecular phenotypic alterations is an attractive strategy for cancer control. We examined whether biomarker profiles could be used for risk assessment and cancer detection in a cohort of Chinese workers occupationally exposed to benzidine and at risk for bladder cancer. METHODS: The cohort consisted of 1788 exposed and 373 nonexposed workers, followed from 1991 through 1997. We assayed urothelial cells from voided urine samples for DNA ploidy (expressed as the 5C-exceeding rate [DNA 5CER]), the bladder tumor-associated antigen p300, and a cytoskeletal protein (G-actin). Workers were stratified into different risk groups (high, moderate, and low risk) at each examination based on a predefined biomarker profile. For workers who developed bladder cancer, tumor risk assessment was analyzed from samples collected 6-12 months before the cancer diagnosis. The associations between risk group and subsequent development of bladder cancer were analyzed by Cox proportional hazards regression analysis and logistic analysis, after adjustment. All statistical tests were two-sided. RESULTS: Twenty-eight bladder cancers were diagnosed in exposed workers and two in nonexposed workers. For risk assessment, DNA 5CER had 87.5% sensitivity, 86.5% specificity, an odds ratio (OR) of 46.2 (95% confidence interval [CI] = 8.1 to 867.0), and a risk ratio (RR) of 16.2 (95% CI = 7.1 to 37.0); p300 had 50.0% sensitivity, 97.9% specificity, an OR of 40.0 (95% CI = 9.0 to 177.8), and an RR of 37.9 (95% CI = 16.8 to 85.3). The risk of developing bladder cancer was 19.6 (95% CI = 8.0 to 47.9) times higher in workers positive for either the DNA 5CER or p300 biomarkers than in workers negative for both biomarkers and 81.4 (95% CI = 33.3 to 199.3) times higher in workers positive for both biomarkers. G-actin was a poor marker of individual risk. CONCLUSIONS: Occupationally exposed workers at risk for bladder cancer can be individually stratified, screened, monitored, and diagnosed based on predefined molecular biomarker profiles.

Mapping of the distribution of significant proteins and proteoglycans in small intestinal submucosa by fluorescence microscopy.

Because small intestine submucosa (SIS) is a bioscaffold for tissue regeneration, we describe a method to analyze the material for growth peptides and for structural molecules. Immunofluorescence methods are described for relative quantification of abundant structural proteins. Additionally, a quantitative technique for comparison of the content of less abundant proteins in SIS was developed using the tyramide signal amplification (TSA) system that is applicable to paraffin-preserved tissue blocks. Frozen sections generally shredded when cut thinly enough to permit entry and washout of reagents. Five micrometer sections cut from paraffin blocks were immunolabeled for collagen, heparan sulfate proteoglycans (HSPG), FGF2, TGFbeta, and VEGF. Images of tissue sections were acquired by a linear image camera and quantified by densitometry after thresholding the signal to minimize nonspecific fluorescence. Immunohistochemistry was used to confirm the immunofluorescence methods. HSPG was widely distributed but concentrated in vessels. FGF2 was distributed diffusely and was associated with fibrous structures. VEGF was distributed mainly around vessels. TGFbeta was barely detectable above background. Collagen fibrils were distinctly present, and with a two-color fluorescence system, the distribution of components relative to collagen can be assessed. The anatomic structure of SIS is likely to play an important role in the regeneration of tissues, and factors in remnant vessels may facilitate penetration of the matrix along these avenues.

Abnormal G-actin content in single prostate cells as a biomarker of prostate cancer.

The distribution of altered G-actin was investigated in prostatic cells obtained by fine needle aspiration (FNA) from 27 excised prostate glands obtained during radical prostatectomy. FNA, which was used to obtain single cells for image analysis, sampled in the region of any nodules and in grossly normal areas of the contralateral lobes. Quantitative fluorescence-image analysis was used to assay the amount of G-actin in individual cells. Abnormal G-actin, a precursor cytoskeletal protein representing cytoskeletal rearrangements accompanying cellular transformation, was associated with the presence of adenocarcinoma in 22 of 27 specimens from the dominant nodule, but only 3 of 20 in the grossly normal specimens (P<.0001). The mean G-actin content of all samples from the dominant nodule was 113.2+/-6.87 and 69.57+/-4.47 from the grossly normal area, the difference being significant at P<.0001. Altered G-actin was not associated with Gleason score (P = .95), grade (P = .26), stage (P = .058), or tumor volume (P = .32), thereby indicating it is a general marker for prostate adenocarcinoma.

Loss of tissue transglutaminase as a biomarker for prostate adenocarcinoma.

BACKGROUND: Additional molecular tissue biomarkers for prostate carcinoma are needed to stratify patients with clinically suspicious findings, such as an elevated prostate specific antigen (PSA) with a negative biopsy, according to risk. METHODS: Prostate tissues from 43 cancer cases and 47 controls with no evidence of cancer were labeled for transglutaminase by immunohistochemistry. Immunoreactivity was quantified using the Autocyte Pathology Workstation. In addition, quantitative fluorescence image analysis was used to compare transglutaminase concentrations in cells obtained by fine-needle aspiration from excised prostates. Loss of gene expression was evaluated by reverse transcriptase-polymerase chain reaction and growth with 5-azacytidine. RESULTS: Visually, benign glands from controls generally expressed tissue transglutaminase, whereas regions with adenocarcinoma generally were negative. With quantitative immunohistochemistry, 41 of 43 adenocarcinoma of the prostate (CaP) cases expressed lower mean percentage areas positive for transglutaminase than did 30 of 30 benign prostatic hyperplasia (BPH) and 17 of 17 prostatitis cases (P < 0.0001; odds ratio [OR], 1577; 95% confidence interval (CI), 74-33, 820; relative risk [RR], 25; 95% CI, 6-95). Quantitative immunofluorescence of 3277 cells collected by FNA from 19 CaP cases and 645 cells from 5 cases of BPH showed that the mean content of transglutaminase was 93 femtograms (fg) for the CaP-derived cells and 138 fg for the BPH cells (P < 0.0001). Receiver operating curve analysis of the immunohistochemistry data showed an optimized threshold produced 95% sensitivity with 100% specificity. Growth of LNCaP cells with 5-azacytidine failed to stimulate transglutaminase expression, suggesting that loss of expression was likely not attributable to promoter methylation. CONCLUSIONS: Measurements of transglutaminase on tissue sections provides additional diagnostic information that is potentially useful for risk assessment of patients with suspicious clinical findings, such as nodules or positive PSA and negative biopsies, without overdetecting disease.

Correctional suicide prevention in the year 2000 and beyond.

Although the problem of suicide in jail and prison historically has been ignored, significant insight has been gained over the past several decades due primarily to increasing litigation. This article provides a brief overview of the progress in research, programming, and policy. The new millennium offers exciting opportunities for continued growth in correctional suicide prevention. The key areas of process research, risk assessment, and public and penological policy changes are reviewed and considered vital for the field's advancement.

A comparison of the microvascular response in the healing wound in the spontaneously hypertensive and non-hypertensive rat.

BACKGROUND: In the Spontaneously Hypertensive Rat (SHR), there is a significantly greater blood flow at the paw but not at the back than in the non-hypertensive Wistar Kyoto (WKY) rat. We wanted to assess the effect of this higher blood flow on wound healing at the paw. MATERIALS AND METHODS: We characterized the microvascular composition of wounds at the back and paw of 9 SHR rats and 10 WKY rats using a quantitative imaging program. Blood flow was compared using laser Doppler technology. RESULTS: The blood flow response to wounding at the back was identical in the SHR and WKY rats. There was an immediate sharp increase in flow at the center of the wound. Blood flow reached a peak at 3 days and then decreased somewhat by day 7, but still remained five-fold higher than the prewound baseline values. There was also a two-fold increase at the back wound perimeter. There were no differences in microvascular composition at the back between the SHR and WKY rats. In contrast, there was an immediate enormous increase in blood flow at paw wound center in the SHR rats. Flow increased to 75 ml/min/100 gm by 24 h then fell back sharply. Blood flow at the paw in the WKY rats changed very little over the 7 days post wounding. At 3 days, the flow was about twice as high in the SHR than in the WKY wound, but, by day 7, flow was similar in the two rat strains. At the SHR wound perimeter, there was a small increase in flow which was sustained through day 7. Although the microvascular composition at the paw wound center was similar in the SHR and WKY rats, there was a notable difference at the paw perimeter. At baseline, there was a slightly greater capillary density in the SHR paw (32 +/- 1 per mm3) than the WKY paw (25 +/- 8 per mm3). At 7 days after wounding, there was a substantial increase in capillary number in the SHR rats (48 +/- 8 per mm3) as compared to baseline (p = 0.05). In contrast, there was no significant difference in capillary number in the WKY paw wound perimeter (20 +/- 3 per mm3) as compared to baseline. CONCLUSIONS: There is a substantial difference in wound blood flow response between the hypertensive and the non-hypertensive rat. At the back, the blood flow effects of wounding are similar, but, at the paw, the SHR rat shows a dramatic transient increase in flow in the early phases of wound healing. There is apparently no capability to upmodulate microvascular resistance in response to increased pressure at this early stage of wound healing. However, within several days, the granulation tissue microvasculature becomes capable of controlling the effects of raised pressure in the SHR rat. In the SHR paw wound perimeter, there are significantly more capillaries than in the WKY rat. It is possible that greater capillary proliferation in the SHR rat results from higher blood flow in the early phase of wounding. The contrast between the WKY rat and the SHR rat serves to further illustrate the complexity of blood flow regulation which occurs during wound healing.

Complexity, retinoid-responsive gene networks, and bladder carcinogenesis.

Carcinogenesis involves inactivation or subversion of the normal controls of proliferation, differentiation, and apoptosis. However, these controls are robust, redundant, and interlinked at the gene expression levels, regulation of mRNA lifetimes, transcription, and recycling of proteins. One of the central systems of control of proliferation, differentiation and apoptosis is retinoid signaling. The hRAR alpha nuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXR alpha regulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network. In this study we showed that simple, cause-effect interpretations in terms of hRAR alpha gene transcription being the central regulatory event would not describe the retinoid-responsive gene network. A set of cultured bladder-derived cells representing different stages of bladder tumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers. Relative gene expression before (constitutive) and after treatment with 10 microM all-trans-retinoic acid (aTRA) was measured for androgen and estrogen receptor; a set of genes involved with retinoid metabolism and action, hRAR alpha nd beta, hRXR alpha and beta CRBP, CRABP I and II; and for signaling genes that are known to be sensitive to retinoic acid, EGFR, cytokine MK, ICAM I and transglutaminase. The phenotype for inhibition of proliferation and for apoptotic response to both aTRA and the synthetic retinoid 4-HPR was determined. Transfection with a CAT-containing plasmid containing an aTRA-sensitive promoter was used to determine if the common retinoic acid responsive element (RARE)-dependent pathway for retinoid regulation of gene expression was active. Each of the genes selected is known from previous studies to react to aTRA in a certain way, either by up- or down-regulation of the message and protein. A complex data set not readily interpretable by simple cause and effect was observed. While all cell lines expressed high levels of the mRNAs for hRXR alpha and beta that were not altered by treatment with exogenous aTRA, constitutive and stimulated responses of the other genes varied widely among the cell lines. For example, CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively. The expression of hRAR alpha, which governs the expression of many retinoid-sensitive genes, was expressed at moderate to high levels in all cell lines, but in some it was sharply upregulated (TCC-Sup, HUC-PC and J82), remained constant (5637 and HUC-BC), or was down-regulated (SCaBER, T24 and RT4). The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa. One line (RT4) was insensitive to either retinoid. Transfection showed very little retinoid-stimulated transfection of the CAT reporter gene with RT4 or HUC-PC. About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3-8 fold with 5637, TCC-Sup cells. In HUC-BC, a G to T point mutation was found at position 606 of the hRAR alpha gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain. These data indicate that retinoid signaling is probably a frequent target of inactivation in bladder carcinogenesis. (ABSTRAC

Visible-light photon migration through myocardium in vivo.

Empirical data between 510 and 590 nm of diffuse reflected light from the pig heart in vivo have shown that myoglobin and cytochrome c absorption peaks with little apparent contribution of red blood cell (RBC) Hb. Monte Carlo simulations of photon migration in tissue were performed to compare the effects of myoglobin and cytochromes with those of blood Hb on photon pathlengths and diffuse reflectance of visible wavelengths (450-600 nm) from the pig heart in vivo. Wavelength dependence of the input parameters, including the transport-corrected scattering coefficients (1.1-1.2 mm(-1)) and the absorption coefficients of blood-free solubilized heart tissue (0.43-1.47 mm(-1)), as well as the absorption coefficients of Hb, were determined by an integrating sphere method and standard spectrophotometry, respectively. The Monte Carlo simulations indicate that in the 510- to 590-nm range the mean path length within the myocardium for diffusely reflected light varies from 1.4 to 1.2 mm, whereas their mean penetration depth within the epicardium is only 330-400 micrometer for blood-free heart tissue. Analysis shows that the blood Hb absorption extrema are only observable between 510 and 590 nm when RBC concentration in tissue is >0.5%. Blood within vessels much larger than capillaries does not contribute significantly to the spectral features, because virtually all light in this spectral range is absorbed during transit through large vessels (>100 micrometer). This analysis suggests that diffuse reflected light in the 510- to 590-nm region will show spectral features uniquely associated with myoglobin and cytochrome c oxygenation states within 400 micrometer of the surface of the heart in situ as long as the capillary RBC concentration remains <0.5%.

A comparison of the cutaneous microvascular properties of the spontaneously hypertensive rat and the Wistar-Kyoto rat.

The Spontaneously Hypertensive rat (SHR) and its non-hypertensive companion strain, the Wistar-Kyoto (WKY) rat, provide an excellent comparative model to permit study of the differential properties of cutaneous microvascular beds. We explored the possibility that chronically elevated vascular pressures in the SHR rat might affect the microvascular constitution of the skin. We measured skin blood flow at the back and at the paw of a group of 20-week-old WKY rats and a contrast group of SHR rats. We then performed skin biopsies at these two locations and used the NIH Image program to count and measure the size of capillaries, arterioles, and venules. We also determined microvascular density as percentage of total tissue area. At basal temperature, skin blood flow was similar in the two rat strains at both the back and paw. Heat induced vasodilatation resulted in a 50% increase in blood flow at the back, reaching the same level in the two rat groups. However, at the paw site, thermal stimulation resulted in significantly greater flow (39.3 +/- 3.1 ml/100 gm tissue per min) in the SHR rats than the WKY rats (28.6 +/- 1.9 ml/100 gm tissue per min, P < 0.05). The ratio of systemic arterial pressure to skin blood flow was computed as an index of vascular resistance to flow. At basal temperature, this index was 50% greater for the SHR rats at both skin sites. At 44 degrees C, the resistance index decreased at both sites in both rat groups but was still approximately 50% higher at the back of the SHR than the WKY rats. In contrast, the resistance index at 44 degrees C at the paw site fell to the same level in both the SHR and WKY rats. There were twice as many capillaries at the back of the WKY rats than at the back of the SHR rats (9.2 +/- 2.0 per mm2 vs. 4.7 +/- 1.2 per mm2, P < 0.05). Expressed as a percentage of total tissue area, the capillary density at the back in the WKY rats was 0.064 +/- 0.010% as compared to 0.034 +/- 0.008% in the SHR rats (P < 0.05). There were five times more arterioles at the paw compared to the back in both rat groups with no significant difference between the groups. We measured the diameter of the lumen and the thickness of the wall of each arteriole and computed their ratio as an index of possible media hypertrophy. There were minimal differences seen in these parameters between the two rat groups at the back and paw sites. The venular density was significantly higher at the paw than at the back in both rat groups with no significant difference between them. Reduced capillary density at the back of the SHR rats may be a developmental adaptation to high blood pressure. Such a reduction in the pathways of blood flow may help account for increased flow resistance at that site, independent of arteriolar vasoconstriction.