Fractal measures of spatial pattern as a heuristic for return rate in vegetative systems.

Measurement of population persistence is a long-standing problem in ecology; in particular, whether it is possible to gain insights into persistence without long time-series. Fractal measurements of spatial patterns, such as the Korcak exponent or boundary dimension, have been proposed as indicators of the persistence of underlying dynamics. Here we explore under what conditions a predictive relationship between fractal measures and persistence exists. We combine theoretical arguments with an aerial snapshot and time series from a long-term study of seagrass. For this form of vegetative growth, we find that the expected relationship between the Korcak exponent and persistence is evident at survey sites where the population return rate can be measured. This highlights a limitation of the use of power-law patch-size distributions and other indicators based on spatial snapshots. Moreover, our numeric simulations show that for a single species and a range of environmental conditions that the Korcak-persistence relationship provides a link between temporal dynamics and spatial pattern; however, this relationship is specific to demographic factors, so we cannot use this methodology to compare between species.

Concentration- and time-dependent genomic changes in the mouse urinary bladder following exposure to arsenate in drinking water for up to 12 weeks.

Inorganic arsenic (As(i)) is a known human bladder carcinogen. The objective of this study was to examine the concentration dependence of the genomic response to As(i) in the urinary bladders of mice. C57BL/6J mice were exposed for 1 or 12 weeks to arsenate in drinking water at concentrations of 0.5, 2, 10, and 50 mg As/l. Urinary bladders were analyzed using gene expression microarrays. A consistent reversal was observed in the direction of gene expression change: from predominantly decreased expression at 1 week to predominantly increased expression at 12 weeks. These results are consistent with evidence from in vitro studies of an acute adaptive response that is suppressed on longer exposure due to downregulation of Fos. Pathways with the highest enrichment in gene expression changes were associated with epithelial-to-mesenchymal transition, inflammation, and proliferation. Benchmark dose (BMD) analysis determined that the lowest median BMD values for pathways were above 5 mg As/l, despite the fact that pathway enrichment was observed at the 0.5 mg As/l exposure concentration. This disparity may result from the nonmonotonic nature of the concentration-responses for the expression changes of a number of genes, as evidenced by the much fewer gene expression changes at 2 mg As/l compared with lower or higher concentrations. Pathway categories with concentration-related gene expression changes included cellular morphogenesis, inflammation, apoptosis/survival, cell cycle control, and DNA damage response. The results of this study provide evidence of a concentration-dependent transition in the mode of action for the subchronic effects of As(i) in mouse bladder cells in the vicinity of 2 mg As(i)/l.

Extrapolating the acute behavioral effects of toluene from 1- to 24-h exposures in rats: roles of dose metric and metabolic and behavioral tolerance.

Recent research on the acute effects of volatile organic compounds suggests that extrapolation from short (∼1 h) to long durations (up to 4 h) may be improved by using estimates of brain toluene concentration (Br[Tol]) instead of cumulative inhaled dose (C × t) as a metric of dose. This study compared predictions of these two dose metrics on the acute behavioral effects of inhaled toluene in rats during exposures up to 24 h in duration. We first evaluated estimates of Br[Tol] with a physiologically based toxicokinetic (PBTK) model for rats intermittently performing an operant task while inhaling toluene for up to 24 h. Exposure longer than 6 h induced P450-mediated metabolism of toluene. Adjusting the corresponding parameters of the PBTK model improved agreement between estimated and observed values of Br[Tol] in the 24-h exposure scenario. Rats were trained to perform a visual signal detection task and were then tested while inhaling toluene (0, 1125, and 1450 ppm for 24 h and 1660 ppm for 21 h). Tests occurred at times yielding equivalent C × t products but different estimates of Br[Tol], and also at 1 and 6 h afterexposure. Effects of toluene were better predicted by Br[Tol] than by C × t. However, even using Br[Tol] as the dose metric (after accounting for metabolic induction), acute dose-effect functions during 24-h exposures were shifted to the right relative to 1-h exposures, indicating that a dynamic behavioral tolerance also developed during prolonged exposure to toluene.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water.

The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites have unique toxicities and relatively greater potency compared to their pentavalent counterparts for many endpoints. In this study, dose-dependency in tissue distribution and urinary excretion for inorganic arsenic and its methylated metabolites was assessed in female C57Bl/6 mice exposed to 0, 0.5, 2, 10 or 50 ppm arsenic (as arsenate, AsV) in their drinking water for 12 weeks. No adverse effects were observed and body weight gain did not differ significantly among groups. Urinary excretion of arsenite monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMAV), and trimethylarsine oxide (TMAO) increased linearly with dose, whereas AsV and monomethylarsonic acid (MMAV) excretion was non-linear with respect to dose. Total tissue arsenic accumulation was greatest in kidney > lung > urinary bladder >>> skin > blood > liver. Monomethyl arsenic (MMA, i.e. MMA(III)+MMAV) was the predominant metabolite in kidney, whereas dimethylarsenic (DMA, i.e., DMA(III)+DMAV) was the predominant metabolite in lung. Urinary bladder tissue had roughly equivalent levels of inorganic arsenic and dimethylarsenic, as did skin. These data indicate that pharmacokinetic models for arsenic metabolism and disposition need to include mechanisms for organ-specific accumulation of some arsenicals and that urinary metabolite profiles are not necessarily reflective of target tissue dosimetry.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in mice following acute oral administration of arsenate.

The relationship of exposure dose and tissue concentration of parent chemical and metabolites is a critical issue in cases where toxicity may be mediated by a metabolite or by parent chemical and metabolite acting together. This has emerged as an issue for inorganic arsenic (iAs), because both its trivalent and pentavalent methylated metabolites have unique toxicities; the methylated trivalent metabolites also exhibit greater potency than trivalent inorganic arsenic (arsenite, As(III)) for some endpoints. In this study, the time-course tissue distributions for iAs and its methylated metabolites were determined in blood, liver, lung, and kidney of female B6C3F1 mice given a single oral dose of 0, 10, or 100 micromol As/kg (sodium arsenate, As(V)). Compared to other organs, blood concentrations of iAs, mono- (MMA), and dimethylated arsenic (DMA) were uniformly lower across both dose levels and time points. Liver and kidney concentrations of iAs were similar at both dose levels and peaked at 1 h post dosing. Inorganic As was the predominant arsenical in liver and kidney up to 1 and 2 h post dosing, with 10 and 100 micromol As/kg, respectively. At later times, DMA was the predominant metabolite in liver and kidney. By 1 h post dosing, concentrations of MMA in kidney were 3- to 4-fold higher compared to other tissues. Peak concentrations of DMA in kidney were achieved at 2 h post dosing for both dose levels. Notably, DMA was the predominant metabolite in lung at all time points following dosing with 10 micromol As/kg. DMA concentration in lung equaled or exceeded that of other tissues from 4 h post dosing onward for both dose levels. These data demonstrate distinct organ-specific differences in the distribution and methylation of iAs and its methylated metabolites after exposure to As(V) that should be considered when investigating mechanisms of arsenic-induced toxicity and carcinogenicity.

A concise review of the toxicity and carcinogenicity of dimethylarsinic acid.

Dimethylarsinic acid (DMA) has been used as a herbicide (cacodylic acid) and is the major metabolite formed after exposure to tri- (arsenite) or pentavalent (arsenate) inorganic arsenic (iAs) via ingestion or inhalation in both humans and rodents. Once viewed simply as a detoxification product of iAs, evidence has accumulated in recent years indicating that DMA itself has unique toxic properties. DMA induces an organ-specific lesion--single strand breaks in DNA--in the lungs of both mice and rats and in human lung cells in vitro. Mechanistic studies have suggested that this damage is due mainly to the peroxyl radical of DMA and production of active oxygen species by pulmonary tissues. Multi-organ initiation-promotion studies have demonstrated that DMA acts as a promotor of urinary bladder, kidney, liver and thyroid gland cancers in rats and as a promotor of lung tumors in mice. Lifetime exposure to DMA in diet or drinking water also causes a dose-dependent increase in urinary bladder tumors in rats, indicating that DMA is a complete carcinogen. These data collectively suggest that DMA plays a role in the carcinogenesis of inorganic arsenic.

Application of modelling techniques to the planning of in vitro arsenic kinetic studies.

A kinetic model describing the hepatic methylation of arsenite [As(III)] was developed on the basis of limited data from in vitro mechanistic studies. The model structure is as follows: sequential enzymic methylation of arsenite to its monomethylated (MMA) and dimethylated (DMA) products by first-order and Michaelis-Menten kinetics, respectively; uncompetitive inhibition of the formation of DMA by As(III); and first-order reversible binding of As(III), MMA and DMA to cytosolic proteins. Numerical sensitivity analysis was used to evaluate systematically the impact of changes in input parameters on model responses. Sensitivity analysis was used to investigate the possibility of designing experiments for robust testing of the uncompetitive inhibition hypothesis, and for further refining the model. Based on the sensitivity analysis, the MMA concentration is the most important response on which to focus. The parameters V(max) and k(i) can be reliably estimated by using the same concentration time-course data at intermediate initial arsenite concentrations of 1--5microM at 30 +/- 5 minutes. K(m) must be estimated independently of V(max), since the two parameters are highly correlated at all times, and the optimal experimental conditions would include lower initial concentrations of arsenite (0.1--0.5microM) and earlier time-points (about 8--18 minutes). The use of initial arsenite concentrations much above 5microM would not yield additional useful information, because the sensitivity coefficients for MMA, protein-bound MMA, DMA and protein-bound DMA tend to become extremely small or exhibit erratic trends. Overall trends in the sensitivity analysis indicated the desirability of performing measurements at times shorter than 60 minutes. This work demonstrates that physiological modelling and sensitivity analysis can be efficient tools for experimental planning and hypothesis testing when applied in the earliest phases of kinetic model development, thus allowing more-efficient and more-directed experimentation, and minimising the use of laboratory animals.

Partnership in nursing care (PINC): the Blackburn model.

Concerns that partnership in care was not really working brought about the need to establish new documentation. A written management plan of care which actively involves the child and family to identify the partnership in care component is essential. Integration of medical and nursing history forms the basic assessment for a jointly managed plan of care. A major culture change needed to be managed to ensure more purposeful and effective communication within the nursing team. The balance of power for nursing care delivery has shifted away from the children's nurse towards the child/parent/carer. The Blackburn PINC model has provided the structures to ensure partnership in care is a reality. Ongoing development and evaluation in response to nursing and parent comments ensures that the process is responsive to current needs.

An immortalized myocyte cell line, HL-1, expresses a functional delta -opioid receptor.

The present study characterizes opioid receptors in an immortalized myocyte cell line, HL-1. Displacement of [(3)H]bremazocine by selective ligands for the mu (mu), delta (delta), and kappa (kappa) receptors revealed that only the delta -selective ligands could fully displace specific [(3)H]bremazocine binding, indicating the presence of only the delta -receptor in these cells. Saturation binding studies with the delta -antagonist naltrindole afforded a B(max)of 32 fmols/mg protein and a K(D)value for [(3)H]naltrindole of 0.46 n M. The binding affinities of various delta ligands for the receptor in HL-1 cell membranes obtained from competition binding assays were similar to those obtained using membranes from a neuroblastomaxglioma cell line, NG108-15. Finally, various delta -agonists were found to stimulate the binding of [(35)S]GTP gamma S, confirming coupling of the cardiac delta -receptor to G-protein. DADLE (D-Ala-D-Leu-enkephalin) was found to be the most efficacious in this assay, stimulating the binding of [(35)S]GTP gamma S to 27% above basal level. The above results indicate that the HL-1 cell line contains a functionally coupled delta -opioid receptor and therefore provides an in vitro model by which to study the direct effects of opioids on cardiac opioid receptors.

Personal fluoride and solvent exposures, and their determinants, in semiconductor manufacturing.

Personal air sampling for fluorides and solvents was done at 35 semiconductor fabrication facilities in the United States. Fluoride compounds were used in etching and cleaning operations, and solvents were used in photoresist and developing operations. All personal solvent and fluoride levels were less than 2 percent of current Occupational Safety and Health Administration (OSHA) standards. Statistical models of the exposure determinants for the target agents found production level, as indicated by number of semiconductor wafer cassettes loaded/unloaded from the target machines or baths, was predictive of fluoride, xylene and 1-methoxy-2-propyl acetate exposures. The percent of fresh air ventilation and the percent of xylene in the photoresist were also significant determinants in the statistical model predicting personal xylene exposure levels.

Dose-dependent effects on tissue distribution and metabolism of dimethylarsinic acid in the mouse after intravenous administration.

Most mammals methylate inorganic arsenic to dimethylarsinic acid (DMA). This organic arsenical causes organ-specific toxicity and is a multi-organ tumor promoter. The objective of this study was to examine whether dose could affect the distribution and metabolism of DMA. Female B6C3F1 mice (3-4/time point) were administered 1.11 or 111 mg/kg of DMA (1 microCi of [14C] or unlabeled) intravenously and killed serially (5-480 min). Blood was separated into plasma and red blood cell fractions and liver, kidney and lung were removed, weighed and homogenized. Tissue samples were oxidized and analyzed for DMA-derived radioactivity. Blood and several organs of the non-radioactive DMA-treated animals were digested in acid and analyzed by hydride generation atomic absorption spectrophotometry for DMA and metabolites. Concentration-time profiles showed a biexponential decrease of DMA-derived radioactivity in all tissues examined. Kidney had the highest concentration (1-20% dose/gm) of radioactivity of all tissues up to 60 min post-administration. Concentration of radioactivity was greater in plasma than red blood cells at 5 and 15 min and then was similar for the remaining time points. A dose-dependent effect on the concentration of radioactivity was observed in the lung. The retention of radioactivity in the lung was altered compared with liver and kidney, with a much longer t1/2beta and a disproportionate increase in area under the curve with increased dose. No methylated or demethylated products of DMA were detected in blood or any organ up to 8 h post-exposure. The dose-dependent distribution of DMA in the lung may have a role in the toxic effects DMA elicits in this organ.

An integrated pharmacokinetic and pharmacodynamic study of arsenite action. 1. Heme oxygenase induction in rats.

Rat heme oxygenase (HO) activity was used as a specific (among forms of arsenic) and sensitive biomarker of effect for orally administered sodium arsenite in rats. Time course studies showed that HO was induced in rat liver from 2 to 48 h in both rat liver and kidney. Hepatic and renal inorganic arsenic (iAs) concentrations were high at times preceding a high degree of HO induction. At times following pronounced HO induction, tissue dimethylarsinic acid concentrations were high. Dose-response studies of arsenite showed substantial HO induction in liver at doses of 30 micromol/kg and higher and in the kidney at doses of 100 micromol/kg and higher. Doses of 10 (in liver) and of 30 micromol/kg (in kidney) sodium arsenite given by gavage did not significantly induce rat HO activity. Speciation of tissue total arsenic into iAs, methylarsonic acid (MMA), and dimethylarsinic acid (DMA) permits us to link tissue iAs and HO enzyme induction. There was a linear relationship between tissue inorganic arsenic (iAs) concentration and tissue HO in individual rats (r(2) = 0.780 in liver and r(2) = 0.797 in kidney). Nonlinear relationships were observed between administered arsenite dose and either liver or kidney iAs concentration. Overall, there was a sublinear relationship between administered arsenite and biological effect in rats. Teratogenesis Carcinog. Mutagen. 19:385-402, 1999. Published 1999 Wiley-Liss, Inc.

Strain-dependent disposition of inorganic arsenic in the mouse.

Recent studies have suggested that polymorphisms in the methylation of inorganic arsenic (iAs) exist in animals and humans. Methylation of iAs is an important step in the elimination of arsenic. The objective of this study was to examine whether there are differences in iAs disposition, and hence methylation, between three strains of mice. Ninety-day-old female mice (strains: C3H/HeNCrlBR, C57BL/6NCrlBR, and B6C3F1/CrlBR) were administered [73As]arsenate or [73As]arsenite orally at dose levels of 0.5 or 5.0 mg As/kg. Another group of mice were administered [73As]arsenate (5.0 mg As/kg) intraperitoneally (i.p.). Disposition of [73As] was assessed by whole-body counting, and analysis of urine, feces and tissues for radioactivity. Urine was analyzed by chromatography for arsenic metabolites. Several strain- and dose-related effects in the disposition of [73As] were observed with both arsenicals. After oral administration, the clearance of [73As]arsenate, measured by whole-body counting, was dependent on the strain. However, because there was no strain dependence on clearance of [73As]arsenate administered i.p., the effect after oral administration may be due to a difference in absorption of arsenate between the strains. With increased oral dose of arsenate and arsenite, the clearance of [73As] was slower and there was higher tissue retention of [73As]. The percentage of metabolites excreted in urine also was affected by the administered dose. With increased dose, the percentage of arsenite and monomethylarsonic acid were significantly increased, and dimethylarsinic acid decreased. However, our results suggest there is no overall difference between these strains of mice with respect to disposition of iAs. A better understanding of the role of phenotype in the disposition and toxicity of iAs would reduce the uncertainty in arsenic risk assessment.

Influence of gender and acetone pretreatment on benzene metabolism in mice exposed by nose-only inhalation.

Benzene (BZ) requires oxidative metabolism catalyzed by cytochrome P-450 2E1 (CYP 2E1) to exert its hematotoxic and genotoxic effects. We previously reported that male mice have a two-fold higher maximum rate of BZ oxidation compared with female mice; this correlates with the greater sensitivity of males to the genotoxic effects of BZ as measured by micronuclei induction and sister chromatid exchanges. The aim of this study was to quantitate levels of BZ metabolites in urine and tissues, and to determine whether the higher maximum rate of BZ oxidation in male mice would be reflected in higher levels of hydroxylated BZ metabolites in tissues and water-soluble metabolites in urine. Male and female B6C3F, mice were exposed to 100 or 600 ppm 14C-BZ by nose-only inhalation for 6 h. An additional group of male mice was pretreated with 1% acetone in drinking water for 8 d prior to exposure to 600 ppm BZ; this group was used to evaluate the effect of induction of CYP 2E1 on urine and tissue levels of BZ and its hydroxylated metabolites. BZ, phenol (PHE), and hydroquinone (HQ) were quantified in blood, liver, and bone marrow during exposure and postexposure, and water-soluble metabolites were analyzed in urine in the 48 h after exposure. Male mice exhibited a higher flux of BZ metabolism through the HQ pathway compared with females after exposure to either 100 ppm BZ (32.0 2.03 vs. 19.8 2.7%) or 600 ppm BZ (14.7 1.42 vs. 7.94 + 0.76%). Acetone pretreatment to induce CYP 2E1 resulted in a significant increase in both the percent and mass of urinary HQ glucuronide and muconic acid in male mice exposed to 600 ppm BZ. This increase was paralleled by three- to fourfold higher steady-state concentrations of PHE and HQ in blood and bone marrow of acetone-pretreated mice compared with untreated mice. These results indicate that the higher maximum rate of BZ metabolism in male mice is paralleled by a greater proportion of the total flux of BZ through the pathway for HQ formation, suggesting that the metabolites formed along this pathway may be responsible for the genotoxicity observed following BZ exposure.

Characterization of plantar verrucae among individuals with human immunodeficiency virus.

Plantar verrucae, caused by human papillomavirus (HPV), are commonly found in patients who have tested positive for the antibodies to human immunodeficiency virus (HIV). A better understanding of the characteristics of plantar verrucae in HIV+ patients in needed. A pilot study was conducted concentrating on three characteristics--the size, the number, and the clinical type--of verrucae present in this population. These parameters were studied in HIV+ and HIV- populations, and they were evaluated in relation to the CD4 levels of HIV+ individuals. The HIV+ individuals presented with plantar verrucae that were larger and more numerous than those found in HIV- individuals. The HIV+ population presented with all three clinical types of plantar verrucae and had significantly more mosaic-type warts than did HIV- individuals. The three characteristics did not correlate with CD4 cell counts, suggesting that the severity and extent of HPV infection do not depend on the level of immunosuppression of the HIV+ patient.

Speech discrimination in the sensorineural hearing loss patient: how is it affected by background noise?

There are patients who have normal hearing below 3,000 Hz and normal speech discrimination who still complain of hearing difficulty, especially when background noise is present. The objective of this study is to document the fact that these individuals have a significant hearing impairment that is not detected with routine testing. We retrospectively reviewed 67 audiograms selected for 50-dB loss or greater at 3, 4, 6, and 8 kHz and speech discrimination scores better than 80%. Patients in this group had also previously undergone speech discrimination testing in the presence of 50-dB calibrated cafeteria noise at the time of the initial audiogram. Identical testing was carried out on 48 control subjects without hearing loss. The speech discrimination scores of the hearing-impaired group were lower than in the control group when tested in a quiet booth (88.2 and 98.2%, respectively). The significant finding was the change in the speech discrimination score when tested in noise. We found that the study group had a 33.1% loss in speech discrimination when tested in the presence of background noise. The control group had only a 5.2% loss in speech discrimination in the presence of the same noisy background; this was statistically different (p = 0.001). Our conclusion is that patients complaining of hearing loss who have normal low- to mid-frequency hearing and good speech discrimination should be tested in the presence of noise to adequately document their degree of impairment. Our findings also support the theories of signal attenuation and secondary auditory pathway distortions as causes of the loss of speech discrimination.

Dose-dependent effects on the disposition of monomethylarsonic acid and dimethylarsinic acid in the mouse after intravenous administration.

The organic arsenicals monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are the primary metabolites of inorganic arsenic, a known human carcinogen. The objective of this study was to examine if dose would affect the excretion and terminal tissue disposition of MMA and DMA in the mouse. 14C-MMA (4.84 and 484 mumol/kg) and -DMA (8.04 and 804 mumol/kg) were administered to female mice via the tail vein. The mice were placed in metabolism cages for collection of urine (1, 2, 4, 8, 12, and 24 h) and feces (24 h). The animals were then sacrificed at 24 h and tissues were removed and analyzed for radioactivity. The urine was also analyzed for parent compound and metabolites. Urinary excretion of MMA- and DMA-derived radioactivity predominated over fecal excretion. Dose did not affect the overall urinary excretion of both compounds. However, fecal excretion was significantly lower in the low-dose MMA-treated animals as opposed to in the high-dose group, whereas in the high-dose DMA-treated group excretion was lower than in the low-dose DMA group. The retention of radioactivity was low (< 2% of dose) and the distribution pattern similar for both compounds, with carcass > liver > kidney > lung. The concentration of radioactivity (% dose/g tissue) was greater in kidney than in liver, lung, and blood for both compounds. The distribution and concentration of MMA-derived radioactivity was significantly greater in the liver and lung of the high-dose group. The MMA-treated animals excreted predominantly MMA in urine and lower amounts of DMA (< 10% of the dose). The percentage excreted as DMA was significantly higher in the low-dose MMA group. In the urine of DMA-treated animals, an unstable metabolite and the parent compound were detected. Overall, it appears the dose of organic arsenical administered has a minimal effect on its excretion and terminal tissue disposition in the mouse. The rapid elimination and low retention of MMA and DMA explain in part their low acute toxicity.

Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium.

pH regulation was studied in fresh explant bovine retinal pigment epithelium-choroid using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and intracellular microelectrodes. Acid recovery was HCO3 dependent, inhibited by apical amiloride and apical or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and required apical and basal Na. Alkali recovery was HCO3 dependent and inhibitable by apical or basal DIDS. Three apical and two basolateral transporters were identified. Four contribute to acid extrusion, i.e., apical Na/H exchange, apical H-lactate cotransport, and apical Na-HCO3 cotransport and basolateral Na-HCO3 cotransport. At least two contribute to alkali extrusion, i.e., apical Na-HCO3 cotransport and a basolateral HCO3-dependent, DIDS-inhibitable mechanism, possibly Na-HCO3 cotransport, Cl/HCO3 exchange, or both. The apical Na-HCO3 cotransporter is electrogenic, carrying net negative charge inward. Basal Cl removal or addition of basal HCO3 caused HCO3- and Cl-dependent alkalinizations, respectively. Apical DIDS increased both responses. These cytosolic pH (pHi) regulatory mechanisms are so tightly coupled that changes in pHi can only occur after two or more of them are inhibited. In addition, these mechanisms help provide pathways for transport of Na and HCO3 across the retinal pigment epithelium between the blood and the distal retina.