INTRODUCTION:Skin cancer (SC) is common in fair skin (FS) at a 1:5 lifetime incidence for nonmelanoma skin cancer. In order to assist clinicians' decisions, a risk intervention technology was developed, which combines a dual-mode machine learning of visual and sonified (pixel to sound) data. The addition of an audio technology enhances malignant features of lesions, increases sensitivity and was previously validated under a prospective clinical setting in FS. In dark skin (DS), although rare by a 10-30 factor, skin cancer is diagnosed at more advanced stages resulting in a delayed diagnosis and affecting life quality and expectancy. It is known as well that SC diagnostic accuracy by machine learning in DS is decreased as compared to FS. The present study tests the use of sonification aided by artificial intelligence algorithms to compare diagnostics of different skin tones. METHODOLOGY:Biopsy-validated smartphone images were diagnosed in a retrospective study by a dual audio-visual convoluted neural network. A total of 60 Fitzpatrick I-III were compared to 72 Fitzpatrick IV-VI. A dichotomous diagnostic output, either malignant or benign, was assessed for sensitivity, specificity and area under the curves (AUCs) for the receiver operating characteristic (ROC) curve. RESULTS:ROC curve analytics indicated an AUC of 0.858 (95% CI: 0.795-0.921) and 0.856 (95% CI: 0.759-0.953) for fair and DS (p = NS). Sensitivity of Fitzpatrick I-III skin and Fitzpatrick IV-VI were 84.4% (71.8-96.9) and 79.6% (63.4-93.8), respectively (p = NS). Specificity of Fitzpatrick I-III skin and Fitzpatrick IV-VI were 84.2% (72.6-95.8) and 85.3% (73.4-97.2), respectively (p = NS). The positive predictive and negative predictive values as well as accuracy (0.817 vs. 0.847) were all within the same range (p = NS). CONCLUSIONS:The results demonstrate that the dual-modality classifier identifies skin cancer of FS and DS similarly well. Sonification of malignant signs of a skin lesion demonstrates promising results, even with smartphone images, which should be considered as a tool to achieve more effective and accessible healthcare.

Purpose Non-melanoma skin cancer (NMSC) is the most frequent keratinocyte-origin skin tumor. It is confirmed that dermoscopy of NMSC confers a diagnostic advantage as compared to visual face-to-face assessment. COVID-19 restrictions diagnostics by telemedicine photos, which are analogous to visual inspection, displaced part of in-person visits. This study evaluated by a dual convolutional neural network (CNN) performance metrics in dermoscopic (DI) versus smartphone-captured images (SI) and tested if artificial intelligence narrows the proclaimed gap in diagnostic accuracy. Methods A CNN that receives a raw image and predicts malignancy, overlaid by a second independent CNN which processes a sonification (image-to-sound mapping) of the original image, were combined into a unified malignancy classifier. All images were histopathology-verified in a comparison between NMSC and benign skin lesions excised as suspected NMSCs. Study criteria outcomes were sensitivity and specificity for the unified output. Results Images acquired by DI ( n = 132 NMSC, n = 33 benign) were compared to SI ( n = 170 NMSC, n = 28 benign). DI and SI analysis metrics resulted in an area under the curve (AUC) of the receiver operator characteristic curve of 0.911 and 0.821, respectively. Accuracy was increased by DI (0.88; CI 81.9–92.4) as compared to SI (0.75; CI 68.1–80.6, p < 0.005). Sensitivity of DI was higher than SI (95.3%, CI 90.4–98.3 vs 75.3%, CI 68.1–81.6, p < 0.001), but not specificity ( p = NS). Conclusion Telemedicine use of smartphone images might result in a substantial decrease in diagnostic performance as compared to dermoscopy, which needs to be considered by both healthcare providers and patients.

Background: Early diagnosis of skin cancer lesions by dermoscopy, the gold standard in dermatological imaging, calls for a diagnostic upscale. The aim of the study was to improve the accuracy of dermoscopic skin cancer diagnosis through use of novel deep learning (DL) algorithms. An additional sonification-derived diagnostic layer was added to the visual classification to increase sensitivity. Methods: Two parallel studies were conducted: a laboratory retrospective study (LABS, n = 482 biopsies) and a non-interventional prospective observational study (OBS, n = 63 biopsies). A training data set of biopsy-verified reports, normal and cancerous skin lesions (n = 3954), were used to develop a DL classifier exploring visual features (System A). The outputs of the classifier were sonified, i.e. data conversion into sound (System B). Derived sound files were analyzed by a second machine learning classifier, either as raw audio (LABS, OBS) or following conversion into spectrograms (LABS) and by image analysis and human heuristics (OBS). The OBS criteria outcomes were System A specificity and System B sensitivity as raw sounds, spectrogram areas or heuristics. Findings: LABS employed dermoscopies, half benign half malignant, and compared the accuracy of Systems A and B. System A algorithm resulted in a ROC AUC of 0.976 (95% CI, 0.965-0.987). Secondary machine learning analysis of raw sound, FFT and Spectrogram ROC curves resulted in AUC's of 0.931 (95% CI 0.881-0.981), 0.90 (95% CI 0.838-0.963) and 0.988 (CI 95% 0.973-1.001), respectively. OBS analysis of raw sound dermoscopies by the secondary machine learning resulted in a ROC AUC of 0.819 (95% CI, 0.7956 to 0.8406). OBS image analysis of AUC for spectrograms displayed a ROC AUC of 0.808 (CI 95% 0.6945 To 0.9208). By applying a heuristic analysis of Systems A and B a sensitivity of 86% and specificity of 91% were derived in the clinical study. Interpretation: Adding a second stage of processing, which includes a deep learning algorithm of sonification and heuristic inspection with machine learning, significantly improves diagnostic accuracy. A combined two-stage system is expected to assist clinical decisions and de-escalate the current trend of over-diagnosis of skin cancer lesions as pathological. (C) 2019 The Author(s). Published by Elsevier B.V.

Background: Skin cancer (SC), especially melanoma, is a growing public health burden. Experimental studies have indicated a potential diagnostic role for deep learning (DL) algorithms in identifying SC at varying sensitivities. Previously, it was demonstrated that diagnostics by dermoscopy are improved by applying an additional sonification (data to sound waves conversion) layer on DL algorithms. The aim of the study was to determine the impact of image quality on accuracy of diagnosis by sonification employing a rudimentary skin magnifier with polarized light (SMP). Methods: Dermoscopy images acquired by SMP were processed by a first deep learning algorithm and sonified. Audio output was further analyzed by a different secondary DL Study criteria outcomes of SMP were specificity and sensitivity, which were further processed by a F2-score, i.e. applying a twice extra weight to sensitivity over positive predictive values. Findings: Patients (n = 73) fulfilling inclusion criteria were referred to biopsy. SMP analysis metrics resulted in a receiver operator characteristic curve AUC's of 0.814 (95% CI, 0.798-0.831). SMP achieved a F2-score sensitivity of 91.7%. specificity of 41.8% and positive predictive value of 57.3%. Diagnosing the same set of patients' lesions by an advanced dermoscope resulted in a F2-score sensitivity of 89.5%, specificity of 57.8% and a positive predictive value of 59.9% ( P = NS). Interpretation: DL processing of dermoscopic images followed by sonification results in an accurate diagnostic output for SMP, implying that the quality of the dermoscope is not the major factor influencing DL diagnosis of skin cancer. Present system might assist all healthcare providers as a feasible computer-assisted detection system. (C) 2019 The Authors. Published by Elsevier B.V.

BACKGROUND Considering increased prevalence of metabolic syndrome in North America, novel therapies that positively affect multiple metabolic risk factors are desirable. Previous studies have demonstrated the pharmacological properties of Korean Red Ginseng of undefined composition to lower blood pressure. OBJECTIVES The present study assessed the acute efficacy of two root fractions–root body and rootlets-of the same batch of Korean Red Ginseng (KRG) having different ginsenoside composition, on blood pressure (BP) and postprandial blood glucose levels. DESIGN Randomized, controlled, double-blind, crossover. METHODS After a 12-hour overnight fast, 13 healthy individuals (Gender:6M:7F, Age:28±10, BMI: 24.1±3kg/m2, SBP: 108±6, DBP: 66±4mmHg) were randomly assigned to receive 3g of either KRG body or rootlet fraction or cornstarch placebo, 40min prior to meal. Part 1: The blood pressure was measured with Ambulatory BP Monitors (90207, SpaceLabs Medical) at 10min intervals for 180min after ingestion of ginseng or placebo. Part 2: Capillary blood samples were taken 60min prior to a meal containing 50g available carbohydrate at which time ginseng or placebo capsules were taken and at 0, 15, 30, 45, 60, 90 and 120min post meal. RESULTS Compared to the placebo, 3g of the rootlet fraction of ginseng root significantly lowered systolic blood pressure at 80min (P<0.05), whereas root body reduced blood glucose iAUC by 19%. CONCLUSION The rootlet fraction of ginseng significantly improved hemodynamic control while the root body fraction had a positive effect on glycemia. This preliminary study supports further investigation of KRG's efficacy on acute and long-term cardiovascular risk factors.

We have previously demonstrated that, among 8 popular ginseng species, cultivated American ginseng (Panax quinquefolius) is one of the most efficacious in lowering postprandial blood glucose. It is not known, however, whether any other batch of cultivated American ginseng would replicate this finding. The present study aimed to assess the glycemia lowering effect of American ginseng from five Ontario farms with varying growing conditions in a controlled multiple cross-over intervention. 12 healthy individuals (5M:7F, age: 27± 2y, BMI: 24 ± 1 kg/m2) were tested on 6 separate occasions, after a 12h overnight fast. Each subject received 9g of American ginseng root from different farms, in random order, or a water control 40 min prior to a 75g oral glucose tolerance test. Venous blood samples were drawn prior to treatment intake and the 75 g glucose load and 15, 30, 45, 60, 90, 120 min after the glucose intake. Comparisons with control showed that ginseng from three farms significantly lowered blood glucose and area under the curve, farm A being the source of the most potent ginseng identified in this study. Overall, American ginseng reduces postprandial glycemia. However different batches do not consistently reproduce the identified glycemia lowering effect. Even among efficacious batches, differential glucose-lowering amplitudes are observed. These variable biologic responses might be explained through compositional differences due to various growing conditions of ginseng. This calls for further identification of the active glycemia-lowering components and subsequent ingredient-based standardization of ginseng.

BACKGROUND Placebo is defined as an inert substance not possessing any biological effect. But this definition may not apply to all placebos. We previously noticed that a cornstarch-placebo decreased the glycemic response to a 75g-oral glucose tolerance test (75g-OGTT) compared with a water-control. Our objective was to investigate the effect of five common placebos on glycemia. METHODS Using a double-blind, randomized, multiple-crossover design, 10 healthy subjects (gender:6M:4F, age:33.1±4y, BMI:27.1±1.7kg/m2) received 7 treatments: 9g glucose-placebo, lactose-placebo, lactulose-placebo, wheat-bran-placebo, and cornstarch-placebo and two water controls. Each treatment was given 40-min before a 75g-OGTT with blood drawn at −40, 0, 15, 30, 45, 60, 90, and 120-min. RESULTS Two-way ANOVA showed a significant effect of treatment(p<0.001) and time (p<0.001) on incremental glycemia, with no interaction. Glucose-placebo significantly reduced AUC by 41±15.4% (p<0.001) and peak glycemia (p<0.005) compared with the mean of the two water-controls. It also reduced AUC (p<0.001) and peak glycemia (p<0.005) significantly compared with the cornstarch, lactulose and wheat-bran placebos. CONCLUSIONS Glucose may not fit the definition of a true placebo. The implication is that the use of glucose as a placebo may lead to underestimation of efficacy in glycemic testing protocols. Travel grant: Inovobiologic, Calgary, AB.

Contrast media (CM) are nephrotoxic and might further worsen renal function in patients with chronic renal failure. L-Arginine, the substrate of nitric oxide, protects kidney function and may improve endothelial function in patients with coronary artery disease.Acute administration of L-arginine in a subset of patients with combined coronary artery disease and impaired kidney function during coronary angiography might prevent superimposed acute renal failure.A double-blind study of patients with mild/moderate chronic renal failure (Cr >1.7 mg/dl) undergoing coronary angiography (meglumine ioxaglate) was conducted. Patients received either L-arginine (300 mg/kg) or placebo and were followed for 48 h. Cardiac hemodynamic parameters, renal function and nitric oxide production were sequentially recorded. RESULTS--PRIMARY AND SECONDARY: Both groups experienced a decrease of creatinine clearance 48 h following the procedure (p < 0.05). Creatinine levels slightly increased following the administration of L-arginine (p < 0.05) but not in the placebo treated group. No changes of systemic and cardiac pressures, total peripheral resistance or cardiac output were recorded within and between the treatment and placebo groups.CM injection causes an impairment of renal function. Addition of intravenous L-arginine during cardiac catheterizations in patients with chronic renal failure does not prevent CM-induced nephrotoxicity and does not affect endothelial dysfunction in the particular population studied by the authors, i.e. patients with coronary artery disease (CAD) of various degrees, or suspicion of CAD and chronic mild renal failure.

Background: Contrast media (CM) are nephrotoxic and might further worsen renal function in patients with chronic renal failure. L-Arginine, the substrate of nitric oxide, protects kidney function and may improve endothelial function in patients with coronary artery disease. Hypothesis: Acute administration of L-arginine in a subset of patients with combined coronary artery disease and impaired kidney function during coronary angiography might prevent superimposed acute renal failure. Methods: A double-blind study of patients with mild/moderate chronic renal failure (Cr >1.7 mg/dl) undergoing coronary angiography (meglumine ioxaglate) was conducted. Patients received either L-arginine (300 mg/kg) or placebo and were followed for 48 h. Cardiac hemodynamic parameters, renal function and nitric oxide production were sequentially recorded. Results – Primary and Secondary: Both groups experienced a decrease of creatinine clearance 48 h following the procedure (p < 0.05). Creatinine levels slightly increased following the administration of L-arginine (p < 0.05) but not in the placebo treated group. No changes of systemic and cardiac pressures, total peripheral resistance or cardiac output were recorded within and between the treatment and placebo groups. Conclusion: CM injection causes an impairment of renal function. Addition of intravenous L-arginine during cardiac catheterizations in patients with chronic renal failure does not prevent CM-induced nephrotoxicity and does not affect endothelial dysfunction in the particular population studied by the authors, i.e. patients with coronary artery disease (CAD) of various degrees, or suspicion of CAD and chronic mild renal failure.

Occlusion has previously been used to treat psoriatic plaques and was shown to improve the condition. We investigated the consequences of applying a mechanical stress, in vitro, on the HaCaT keratinocyte cell line. A mechanical load applied to cells can be mimicked by a hyperosmotic stimulus. Exposure of HaCaT keratinocytes to different hyperosmotic solutions (final osmolarity in the range 350-600 mOsm, produced by sucrose addition) resulted in an inhibition of cell proliferation after 96 h of treatment. As keratinocyte maturation is regulated by calcium levels, we measured hyperosmotic-stimulus-induced changes of intracellular calcium ([Ca2+]i) by single-cell image analysis employing FURA-2/AM. The hyperosmotic stimulus produced a rapid transient 2.6-fold elevation of [Ca2+]i followed by a gradual decay to the basal level. The transients originated from extracellular as well as from intracellular calcium pools and did not respond to voltage-sensitive calcium channel blockers. The hyperosmotic stimulus was shown to increase the cellular expression of involucrin, a differentiation marker, following 72 h of incubation, as measured by flow cytometry. Treatment of cells with the [Ca2+]i chelator BAPTA/AM almost completely blocked the [Ca2+]i elevation, but did not alter cellular growth or the induction of differentiation observed after hyperosmotic stimulus. It is suggested that treatment of keratinocytes with hyperosmotic stimulus can induce short-time effects (calcium transients) as well as long-term cellular maturation.

Standard methods for accurate intraoperative measurement of neuromuscular block are either expensive or inconvenient and are not used widely. We have evaluated a new method of monitoring neuromuscular block using a low-frequency microphone. The method is based on the phenomenon of low-frequency sound emission by contracting skeletal muscle. Acoustic monitoring (MIC) with an air-coupled microphone was used to evaluate intraoperative neuromuscular block in 25 anaesthetized patients. The MIC recorded the response of the adductor pollicis muscle to supramaximal electrical stimulation of the ulnar nerve with train-of-four stimuli. The ratios of the first response (TI) to control (TC) were used for evaluation. Data obtained from the MIC were compared with simultaneous recordings, from the same hand, of mechanomyography (FDT), electromyography (EMG) and accelerography (ACC). Throughout the operative procedure, TI/TC ratios of the acoustic method correlated with the three reference devices: FDT, 12 patients, 262 data sets, r = 0.86, bias (%MIC-%FDT) = mean -5.3 (SD 19.6)%; EMG, 18 patients, 490 data sets, r = 0.85, bias (%MIC-%EMG) = -0.39 (20.29)%; and ACC, 13 patients, 328 data sets, r = 0.91, bias (%MIC-%ACC) = -3.0 (15.6)%. We conclude that monitoring intraoperative neuromuscular block by a microphone which transduces low-frequency muscle sounds is clinically feasible.

Cartilage is exposed to mechanical loads, generating at the level of single chondrocytes a hyperosmotic stimulus (HOS). The direct effect of HOS on second messenger pathways in avian chondrocytes was evaluated by fluorimetric and image analysis techniques. HOS caused an immediate intracellular acidification of 0.07 +/- 0.02 pH units (n = 7), followed by an initial pH recovery rate of 0.033 +/- 0.04 pH units/min towards the pre-stimulus baseline values. Concomitantly, the intracellular calcium ([Ca2+]i) responded with a transient rise from baseline value of 84.7 +/- 7.4 nM to peak level of 403.1 +/- 51.0 nM (n = 16, p < 0.001). The calcium response was abolished by two calmodulin inhibitors chlorpromazine and W-7. Since these inhibitors are known to be specific ligands of a S-100 protein, its intracellular staining was determined following HOS. The amount of immunodetectable S-100 protein was significantly increased following exposure to HOS (p < 0.05), and did not require an increase of [Ca2+]i. It appears that compression of cartilage is transduced into HOS of chondrocytes, and further elicits its effects through transient intracellular elevation of protons and calcium ions accompanied by increased staining of S-100 protein.

1. The effects of hyperosmotic stress on cytosolic calcium concentration ([Ca2+]i) were studied by ratio image analysis in single cells of an osteoblast‐like bone cell line (RCJ 1.20) loaded with fura‐2 AM. 2. The ratio (340 nm/380 nm) of steady‐state [Ca2+]i in resting osteoblasts kept in Hepes‐buffered medium was 0.82 +/‐ 0.04. A hyperosmotic stimulus (200 mosmol l‐1 sucrose) produced a [Ca2+]i transient with a peak ratio of 1.28 +/‐ 0.09, which decayed with an apparent half‐life (t1/2) of 42.7 +/‐ 2.6 s. 3. The hyperosmotically induced [Ca2+]i transients were insensitive to verapamil, diltiazem or nifedipine, which excludes the involvement of dihydropyridine‐sensitive Ca2+ channels in the process. Non‐specific Ca2+ channel blockers (Mn2+, Ni2+, La3+ or Gd3+) partially abolished the hyperosmotically induced [Ca2+]i elevation, indicating the contribution of extracellular Ca2+ influx. 4. A hyperosmotic stimulus applied in Ca(2+)‐free medium (0.5 mM EGTA) lowered the [Ca2+]i peak to a ratio of 0.96 +/‐ 0.08 (P < 0.001) compared with a Ca(2+)‐containing medium. This suggests that the [Ca2+]i increase is due to extracellular influx, as well as release from an intracellular Ca2+ pool. 5. Application of thapsigargin (0.5 microM), a specific inhibitor of endoplasmic reticulum Ca(2+)‐ATPase, in Ca(2+)‐free medium caused transient [Ca2+]i elevation to peak ratios of 1.33 +/‐ 0.09, and completely abolished the [Ca2+]i response to a hyperosmotic stimulus. This implies the existence of a thapsigargin‐sensitive intracellular pool of Ca2+ that is mobilized by hyperosmotic stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

RATIONALE AND OBJECTIVES:Radiologic contrast media (CM) are hyperosmotic compounds injected undiluted into a patient's blood, in which they contact endothelial cells. For some types of cultured cells, the application of a hyperosmotic stimulus may cause intracellular pH (pHi) acidification that is related to the extent of hyperosmolality and that ultimately influences cellular function. Accordingly, endothelial and kidney cells, two types of cells known to be exposed to CM effects, were treated at relevant iodine concentrations with various CM (320-1500 mOsm) to determine whether cell exposure to CM can disturb the pH(i) and to examine the contribution of CM to cellular cytotoxicity. METHODS:Ionic (n = 3) and nonionic (n = 3) CM were compared. Changes in the pH(i) of human vascular endothelial and kidney cell lines were monitored by use of a pH-sensitive fluorescent dye (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester). The viability of cells treated with CM was determined by measuring the reduction of a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenlytetrazolium bromide) to violet formazan, a reaction that requires the activity of mitochondrial dehydrogenase; this measurement was made with a microplate reader. RESULTS:The pH(i) of endothelial and kidney cells exposed to 40-60 mg of iodine per milliliter of CM showed acidification (approximately 0.2 pH unit). Within minutes, gradual pH(i) alkalinization to baseline values occurred. The return to baseline values was slower with ionic compounds than with nonionic CM (P < 0.001). Nonionic agents caused less cellular damage than did ionic CM. CONCLUSIONS:The pH(i) is involved in the immediate intracellular transduction of CM effects in vitro. The exposure of cells to ionic CM is more detrimental than is exposure to nonionic CM, as demonstrated by disturbances in the cytosolic pH and by long-term effects on cell viability.

1. Mechanical loading of cartilaginous tissue generates an increase in the concentration of cations in the extracellular matrix. This includes a decrease of the extracellular pH (pHo), which is known to affect the intracellular pH (pHi), thereby modifying the intracellular metabolism. Thus, the regulation of pHi is essential for the physiological function of cartilage. The fluorescent pH‐sensitive dye 2',7'‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed in order to assess the mechanisms responsible for control of the pHi in an embryonic avian chondrocyte cell suspension. 2. Steady‐state pHi in the absence of physiological HCO3‐ was 7.15 +/‐ 0.01 pH units as compared to a pHi of 6.94 +/‐ 0.02 pH units in its presence (P < 0.01). The intrinsic buffering power of chondrocytes (beta i) was 38.9 mM/pH unit and the total buffering capacity (beta T) was 65.8 mM/pH unit. 3. Cells maintained in a Hepes‐buffered solution were exposed to an intracellular acid load by the NH4+ prepulse technique (20 mM NH4Cl). The initial rate of pHi recovery was 0.106 pH units/min (n = 18). Amiloride (0.33 mM), an inhibitor of the Na(+)‐H+ exchanger, or replacement of external sodium [Na+]o with choline induced a 60% inhibition of the recovery rate, indicating a predominant involvement of this antiporter in the response to intracellular acidification. 4. H(+)‐ATPase inhibitors (oligomycin 20 micrograms/ml; N,N;‐dicyclohexylcarbodiimide (DCC), 0.5 mM; N‐ethylmaleimide (NEM), 0.25 mM) and iodomycin (2 mM), a metabolic cell suppressor, reduced acid extrusion by 25% as measured by the NH4Cl prepulse in Hepes‐bathed cells. 5. Chondrocytes transferred from a Hepes‐buffered solution to a 5% CO2‐25 mM HCO3‐ medium (HCO3‐ solution) underwent a pHi decrease of approximately 0.20 pH units, followed by a regulatory alkalinizing response of 0.118 pH units/min. The Na(+)‐H+ exchanger was responsible for only 15% of this alkalinization (amiloride, 0.33 mM), in contrast to its primary role in HCO(3‐)‐free solution. 6. The activity of a Na(+)‐dependent Cl(‐)‐HCO3‐ exchanger in physiological HCO3‐ solution was estimated by addition of the inhibitors 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid (SITS; 0.5 mM) or diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS; 100 microM) and by the suspensions of chondrocytes in a Na(+)‐free solution. Acidification performed under these conditions resulted in a 45% inhibition of the recovery rate as compared to control rates.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The hyperosmotic activation of the Na(+)-H+ exchanger was studied in an osteoblast-like rat cell line (RCJ 1.20). The activation was monitored by recording the intracellular pH (pHi) changes employing double excitation of the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). 2. Exposure of the cells to a hyperosmotic HCO(3-)-free medium at 37 degrees C produced an initial cytosolic acidification of 0.05 pH units followed by a lag period and an alkalinization overshoot of about 0.2 pH units, without a concomitant change of the free cytosolic calcium [Ca2+]i by the use of Fura-2 calcium-sensitive probes. This response was completely inhibited by amiloride (0.33 mM) or by Na+ depletion from the external medium and insensitive to the extracellular Cl- replacement, indicating the involvement of a Na(+)-H+ exchanger in the hyperosmotic response. 3. Hyperosmotic stimuli (200 moSM sucrose) applied in the temperature range of 17-37 degrees C demonstrated a shortening of the lag period preceding alkalinization and an increased rate of proton extrusion upon temperature elevation. The biochemical reaction underlying the lag period and the proton extrusion resulted in apparent activation energies of 19 and 29 kcal mol-1, respectively, as calculated from the appropriate Arrhenius plots. 4. Stimulation of the exchanger under isosmotic conditions by 25 nM 4 beta-phorbol 12-myristate 13-acetate (PMA) and 0.1 mM vanadate resulted in an amiloride-sensitive pHi increase of about 0.08 pH units. The hyperosmotic stress was additive to the stimulatory effects of these agents, suggesting an independent hyperosmotic activation pathway. 5. The hyperosmotic activation of the Na(+)-H+ exchanger was independent of cAMP, cGMP, cytosolic Ca2+ and protein kinase C. Thus, none of the classical transduction mechanisms seem to be involved directly in the hyperosmotic activation of the antiporter. 6. The pHi response induced by the hyperosmotic stress was abolished by two calmodulin inhibitors, W-7 and chlorpromazine (50% inhibition, Ki at 28 and 20 microM, respectively), 20 microM cytochalasin B, but not by 10 microM colchicine. The results suggest the involvement of actin and calmodulin-like structural elements of the cytoskeleton in the transduction process leading to the activation of the Na(+)-H+ exchanger.

Activation of the Na + /H + exchanger following isosmotic and hyperosmotic stimuli was investigated in an osteoblast cell line (RCJ 1.20). The pH dependence of the transporter activity was studied under conditions of abolished proton gradient (pH i = PH*) across the membrane. The isotonic response is Na + * dependent, increases towards higher pH. values, displaying a sigmoidal dependence on pH 1 ** (Hill coefficient ≈ 1.8) and is controlled by pH*. The greater than first order dependence of pH suggest that H + * inhibits the exchange beyond the rate expected from competition with the Na + * alone. This may be due to the existence of an external H + regulatory site with a negative cooperative effect on the intra‐ or extracellular transport site. The hyperosmotic activation is Na + * independent, parallels the sigmoidal pH dependence of the isosmotic stimulus (Hill coefficient ≈ 2.0) and is mediated through an increase of the V max without a change in the intracellular proton sensitivity.