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Introduction and Objectives: Advanced analysis of the morphological features of the photoplethysmographic (PPG) waveform may provide greater understanding of mechanisms of action of photobiomodulation (PBM). Photobiomodulation is a non-ionizing, red to near-infrared irradiation shown to induce peripheral vasodilatation, promote wound healing, and reduce pain. Using laser Doppler flowmetry combined with thermal imaging we found previously in a clinical study that PBM stimulates microcirculatory blood flow and that baseline palm skin temperature determines, at least in part, why some individuals respond favorably to PBM while others do not. "Responders" (n = 12) had a skin temperature range of 33°C-37.5°C, while "non-responders" (n = 8) had "cold" or "hot" skin temperature (<33°C or >37.5°C respectively). The continuous PPG signals recorded from the index fingers of both hands in the original clinical study were subjected to advanced post-acquisitional analysis in the current study, aiming to identify morphological features that may improve the accuracy of discrimination between potential responders and non-responders to PBM. Methods: The PPG signals were detrended by subtracting the lower envelope from the raw signal. The Root Mean Square (RMS) and Entropy features were extracted as were two additional morphological features -- Smoothness and number of local extrema per PPG beat (#Extrema). These describe the signal jaggedness and were developed specifically for this study. The Wilcoxon test was used for paired comparisons. Correlations were determined by the Spearman correlation test (r s ). Results: The PPG waveforms of responders to PBM had increased amplitude and decreased jaggedness (Baseline vs. 10' post-irradiation: Entropy, 5.0 ± 1.3 vs. 3.9 ± 1.1, p = 0.012; #Extrema, 4.0 ± 1.1 vs. 3.0 ± 1.6, p = 0.009; RMS, 1.6 ± 0.9 vs. 2.3 ± 1.2, p = 0.004; Smoothness, 0.10 ± 0.05 vs. 0.19 ± 0.16, p = 0.016). In addition, unilateral irradiation resulted in a bilateral response, although the response of the contralateral, non-irradiated hand was shorter in duration and lower in magnitude. Although subjects with 'cold,' or 'hot,' baseline skin temperature appeared to have morphologically distinct PPG waveforms, representing vasoconstriction and vasodilatation, these were not affected by PBM irradiation. Conclusion: This pilot study indicates that post-acquisitional analysis of morphological features of the PPG waveform provides new measures for the exploration of microcirculation responsiveness to PBM., Competing Interests: Author BG was employed by Yazmonit Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ovadia-Blechman, Hauptman, Rabin, Wiezman, Hoffer, Gertz, Gavish and Gavish.)

Background: The role of pulse pressure (PP) 'widening' at older and younger age as a cardiovascular risk factor is still controversial. Mean PP, as determined from repeated blood pressure (BP) readings, can be expressed as a sum of two components: 'elastic PP' (elPP) and 'stiffening PP' (stPP) associated, respectively, with stiffness at the diastole and its relative change during the systole. We investigated the association of 24-h ambulatory PP, elPP, and stPP ('PP variables') with mortality and composite cardiovascular events in different age classes., Method: Longitudinal population-based cohort study of adults with baseline observations that included 24-h ambulatory BP. Age classes were age 40 or less, 40-50, 50-60, 60-70, and over 70 years. Co-primary endpoints were total mortality and composite cardiovascular events. The relative risk expressed by hazard ratio per 1SD increase for each of the PP variables was calculated from multivariable-adjusted Cox regression models., Results: The 11 848 participants from 13 cohorts (age 53 ± 16 years, 50% men) were followed for up for 13.7 ± 6.7 years. A total of 2946 participants died (18.1 per 1000 person-years) and 2093 experienced a fatal or nonfatal cardiovascular event (12.9 per 1000 person-years). Mean PP, elPP, and stPP were, respectively, 49.7, 43.5, and 6.2 mmHg, and elPP and stPP were uncorrelated ( r  = -0.07). At age 50-60 years, all PP variables displayed association with risk for almost all outcomes. From age over 60 years to age over 70 years, hazard ratios of of PP and elPP were similar and decreased gradually but differently for pulse rate lower than or higher than 70 bpm, whereas stPP lacked predictive power in most cases. For age 40 years or less, elPP showed protective power for coronary events, whereas stPP and PP predicted stroke events. Adjusted and unadjusted hazard ratio variations were similar over the entire age range., Conclusion: This study provides a new basis for associating PP components with outcome and arterial properties in different age groups and at different pulse rates for both old and young age. The similarity between adjusted and unadjusted hazard ratios supports the clinical usefulness of PP components but further studies are needed to assess the prognostic significance of the PP components, especially at the young age., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Pulse pressure (PP) reflects the age-related stiffening of the central arteries, but no study addressed the management of the PP-related risk over the human lifespan., Methods: In 4,663 young (18-49 years) and 7,185 older adults (≥50 years), brachial PP was recorded over 24 hours. Total mortality and all major cardiovascular events (MACEs) combined were coprimary endpoints. Cardiovascular death, coronary events, and stroke were secondary endpoints., Results: In young adults (median follow-up, 14.1 years; mean PP, 45.1 mm Hg), greater PP was not associated with absolute risk; the endpoint rates were ≤2.01 per 1,000 person-years. The adjusted hazard ratios expressed per 10-mm Hg PP increments were less than unity (P ≤ 0.027) for MACE (0.67; 95% confidence interval [CI], 0.47-0.96) and cardiovascular death (0.33; 95% CI, 0.11-0.75). In older adults (median follow-up, 13.1 years; mean PP, 52.7 mm Hg), the endpoint rates, expressing absolute risk, ranged from 22.5 to 45.4 per 1,000 person-years and the adjusted hazard ratios, reflecting relative risk, from 1.09 to 1.54 (P < 0.0001). The PP-related relative risks of death, MACE, and stroke decreased >3-fold from age 55 to 75 years, whereas absolute risk rose by a factor 3., Conclusions: From 50 years onwards, the PP-related relative risk decreases, whereas absolute risk increases. From a lifecourse perspective, young adulthood provides a window of opportunity to manage risk factors and prevent target organ damage as forerunner of premature death and MACE. In older adults, treatment should address absolute risk, thereby extending life in years and quality., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd.)

Objective: Twenty-four-hour ambulatory pulse pressure (PP) is a powerful predictor of outcome. We attempted to apply the recently described PP components, an elastic (elPP), and systolic stiffening (stPP) components from 24-h ambulatory blood pressure (BP) monitoring (AMBP), and examine their influence on outcome in the Ohasama study population., Design and Methods: Included were participants of the Ohasama study without history of cardiovascular disease (CVD), who were followed-up for total and CVD mortality, and for stroke morbidity. The PP components were derived from 24-h SBP and DBP using a model based on the nonlinear pressure--volume relationship in arteries expressing pressure stiffness relationship. Outcome predictive power was estimated by Cox regression models; hazard ratio with 95% confidence interval (CI), applied to elPP, and stPP, adjusted for age, sex, BMI, smoking, alcohol drinking, diabetes mellitus, total cholesterol, antihypertensive treatment, and mean arterial pressure (MAP), whenever appropriate., Results: Of 1745 participants (age 61.4 ± 11.6, 65% women), 580 died, 212 of CVD, and 290 experienced a stroke during 17 follow-up years. PP was strongly correlated with elPP (r = 0.89) and less so with stPP (r = 0.58), and the correlation between the two components was weak (r = 0.15). After the adjustment, hazard ratio of PP per 1 SD increment for total mortality, CVD mortality, and stroke morbidity were 1.095 (95% CI 0.973-1.232), 1.207 (1.000-1.456), and 0.983 (0.829-1.166), respectively. Corresponding hazard ratios and 95% CIs were nonsignificant for elPP, and stPP. However, among participants with median pulse rate 68.5 bpm or less (median, n = 872), total (327 deaths) and CVD (131 deaths) mortality were predicted by elPP (per 1 SD increment), hazard ratio 1.231 (95% CI, 1.082-1.401), and 1.294 (95% CI, 1.069-1.566), respectively. In the subgroup of treated participants with hypertension and pulse rate 68.5 or less bpm (n = 309), total (177 deaths) and CVD (77 deaths) mortality were predicted by elPP, hazard ratio of 1.357 (95% CI, 1.131-1.628), and 1.417 (95% CI, 1.092-1.839), respectively. Stroke morbidity was not predicted by either PP or the PP components., Conclusion: In a rural Japanese population, elPP but not stPP was predictive of total and CVD mortality even when adjusted for MAP and conventional risk factors in the subpopulation with slower pulse rate. This was mostly among the treated hypertensive patients.

Background: Ambulatory pulse pressure (PP) a well known predictor of mortality, is widely believed to be a marker for arterial stiffness. However stiffness itself is pressure-dependent., Objectives: Developing a model-based method for splitting PP into two components expressing the contribution of a pressure-independent stiffness ('elastic', elPP) and the pressure-dependence of stiffness ('stiffening', stPP), and investigating their predictive power for all-cause mortality., Methods: Deriving quantitative expressions for elPP and stPP assuming an exponential pressure-volume relationship in arteries, calculated from ambulatory blood pressure (BP) data and estimate standardized hazard ratios with Cox proportional hazards regression in selected patient groups., Results: Ambulatory BP records of 1999 consecutive hypertensive patients, of whom 103 died from all causes within 5 years, were analyzed. PP, elPP, stPP and stPP/elPP (PP variables) (mean ± SD) were 60 ± 14, 50 ± 10, 10 ± 8 and 0.20 ± 0.14 mmHg, respectively. elPP and stPP were weakly correlated (r = 0.21). Predictive power was found for patients with heart rate less than 70: mean [95% confidence interval] (P value) hazard ratio of PP, stPP and stPP/elPP (adjusted) were 1.48 [1.13-1.95] (P = 0.005), 1.58 [1.20-2.09] (P = 0.001) and 1.78 [1.25-2.52] (P = 0.001), respectively. The elPP (83% of PP) did not show predictive power. None of the PP variables displayed predictive power for the higher-heart-rate subgroup., Conclusion: Ambulatory PP components provide a novel and clinically valuable tool bridging between PP and arterial properties. Replacing PP by its components, taken as independent variables, may improve predictive power. The prognostic significance of the PP for all-cause mortality in elderly hypertensive patients with lower heart rates is dominated by its smaller component that quantifies arterial stiffening with pressure.

Background: The peripheral microcirculation supplies fresh blood to the small blood vessels, providing oxygen and nutrients to the tissues, removing waste, and maintaining normal homeostatic conditions. The goal of this study was to characterize the response of the peripheral microcirculation, in terms of blood flow and tissue oxygenation variables, to gravity-induced changes., Methods: The study included 20 healthy volunteers and the experiment involved monitoring central and peripheral variables with the right hand positioned at different heights. These positions correspond to various gravitational levels. Peripheral perfusion and oxygenation were monitored using a laser Doppler flowmeter, photoplethysmograph, and transcutaneous oxygen tension monitor. Local blood pressure and respiration rate were also measured., Findings: At the heart level, tissue oxygenation displayed a nadir, while capillary flow displayed a peak. Similar but weaker changes were observed at the control hand. In contrast, the photoplethysmograph's amplitude strongly decreased upon reducing the arm position below heart level. Both systolic and diastolic pressures decreased linearly between the highest to lowest arm position., Interpretation: The results may reflect peripheral compensation mechanisms, as well as an interaction between the central and peripheral cardiovascular systems, in response to local changes in blood pressure. The observed dependence of the oxygenation pattern on height could lead to important new insights for the diagnosis and treatment of problems in the regulation of tissue perfusion., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Blood pressure variability ratio (BPVR)(derived from within-subject SD of 24-hour ambulatory blood pressure [BP]) predicts all-cause mortality independent of BP and has a similar prognostic ability to ambulatory arterial stiffness (AASI). Whether BPVR, and AASI, offer prognostic information beyond measurements of arterial stiffness at a given pressure, as indexed by pulse wave velocity (PWV), is not known., Methods: We assessed whether BPVR and AASI were associated with indices of subclinical organ damage (TOD) [estimated glomerular filtration rate (eGFR), left ventricular mass index, early-to-late transmitral velocity (E/A), carotid intima-media thickness (IMT)] independent of BP, and whether BPVR-TOD and AASI-TOD relations were independent of PWV (applanation tonometry) in 772 randomly selected participants from an urban, developing community. AASI was derived from 24-hour diastolic BP vs. systolic BP standard linear regression., Results: On bivariate analyses, BPVR, AASI, and PWV were correlated with all indices of TOD (P < 0.0005). However, after adjustments for potential confounders including age and 24-hour mean BP, BPVR, and PWV (P < 0.005 to P < 0.0001), but not AASI (P > 0.25), were independently associated with eGFR, but not other indices of TOD. Importantly, the BPVR-eGFR relation was independent of BP variability (P < 0.005) and PWV (P < 0.001)., Conclusions: BPVR was negatively associated with eGFR independent of mean BP, BP variability, and PWV. Therefore, in the prediction of cardiovascular risk, measurements of arterial stiffening (BPVR) may provide information beyond the impact of arterial stiffness., (© American Journal of Hypertension, Ltd 2017. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Vasomotion (rhythmic changes in arteriolar diameter) is believed to enhance tissue perfusion at low oxygenation levels. We hypothesized that slow breathing and vasomotion may correlate temporally ("coupling"), especially at low oxygenation levels. We paced down spontaneous breathing to about 5 or 6breaths/min in 14 healthy subjects using device-guided breathing (DGB), and continuously monitored respiration, transcutaneous oxygen pressure ("oxygenation"), and skin capillary blood flow ("microflow") using a laser Doppler flowmeter. The coupling was expressed by cross-correlation calculated in 1-min time windows. Our main results illustrated that: (1) coupling increased gradually upon slowing breathing down in a subgroup, in which initial oxygenation was lower than a threshold of 30mmHg (0.3±0.2 vs. 0.07±0.2, P<10 -6 ); (2) during DGB changes in oxygenation elicited opposite (relative) changes in microflow, with 4-fold higher sensitivity for low initial oxygenation relative to high (regression slope -0.094±0.010mmHg -1 vs. -0.020±0.002mmHg -1 , P<10 -6 ); (3) at low initial oxygenation, we observed larger coupling and (relative) microflow changes in younger subjects, and greater oxygenation changes in females (P<10 -6 for all); (4) pulse pressure changes from before to after DGB were reduced by increased oxygenation changes during DGB (-5.5±7.4mmHg, r=-0.73, P<0.001). In conclusion, the present methodology can provide the variation trend of respiration-vasomotion coupling during DGB that may characterize microcirculation behavior at tissue oxygenation below a measurable threshold. The potential association of these trends and thresholds with pathologies or specific conditions of the cardiopulmonary system, and the possible role played by the neural sympathetic activity in that coupling, deserve further studies., (Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.)

Interest in arterial stiffness has been fueled by the scientific and clinical implications of its "vicious cycle" relationship with aging and systolic blood pressure. In physical terms, stiffness is the slope of the relationship between an artery's distending pressure and its cross-sectional area or volume. Pulse wave velocity (PWV, in m/s), the most common arterial stiffness indicator, is usually measured by the foot-to-foot time and distance method and is proportional to [stiffness × area (or volume)]1/2 at a given pressure. Its intrinsic pressure dependency and other flaws in current PWV methods limit its utility. In contrast, the arterial stiffness-arterial pressure relationship is near-linear, with a slope β, the exponent of the curvilinear arterial pressure-arterial volume relationship. The concept of arterial stiffening is related to β and describes a more functionally relevant aspect of arterial behavior: the change in stiffness for a given change in pressure. Arterial stiffening can be estimated from the variability of within-individual BP measurements (24-h ambulatory, home BP, or BP measured at different arm heights) and can be expressed as the pulse stiffening ratio (PSR) = [systolic stiffness]/[diastolic stiffness] or the ambulatory arterial stiffness index (AASI or its symmetric form, sAASI). High arterial stiffness (PWV) and stiffening (β, stiffness index, cardio-ankle vascular index, AASI, and PSR) are associated with increased cardiovascular disease risk, but it remains unclear whether these indicators are useful in improving medical care quality; the standard of care remains stringent BP control.

Background: Local blood pressure (BP) changes induced by arm tilting may influence pressure wave transmission and reflection. We investigated the effects of upper-limb tilting on radial augmentation index (rAIx) and related central measures [aortic augmentation index (aAIx)]., Methods: In 45 volunteers (age 49 ± 19 years), supine brachial BP and radial artery waveforms were obtained by applanation tonometry with the dominant arm stretched and gently supported in three different positions: at the heart level, with the BP cuff 15 cm above heart level (approximately +30°), and 15 cm below heart level (-30°)., Results: Brachial SBP/DBP was 120/68 ± 17/8 mmHg. Mean arterial pressure changed predictably with arm tilting (99 ± 12 mmHg at -30°, 88 ± 10 mmHg at 0°, 77 ± 11 mmHg at +30°, all P < 0.001). rAIx decreased at -30° (69 ± 22%), and increased at +30° (93 ± 20%) compared with 0° (82 ± 20%, all P less than 0.001). Changes in rAIx (value at +30° minus value at -30°) showed an inverse relationship with age (r = -0.32, P = 0.03). Heart rate, BP and rAIx did not change in the contralateral arm, which was held at the heart level during the examination. aAIx followed the same pattern as rAIx (123 ± 27% at -30°, 144 ± 33% at +30°, 136 ± 31% at 0°, all P less than 0.001); changes in rAIx and aAIx were strongly related each other (r = 0.82, P < 0.001)., Conclusion: Acute gravitational upper-limb BP changes generate opposite, profound changes in rAIx, and major artifactual changes in aAIx. These findings provide a rationale for recommending to keep the upper limb at the heart level during radial waveform assessment.

Objective: The linear relationship between blood pressure (BP) and heart rate (or period) over 24 h has been suggested to be a marker for neural regulation of the circadian variations in BP and heart rate. We investigated the predictive power of indices defined by the ratio between BP and heart period variability that is a known expression for such a relationship., Methods: We analysed BP and heart rate data obtained by standard 24-h BP monitoring in 1246 hypertensive patients, 76 of whom died from all causes during 5-year follow-up. SBP, DBP and heart period (T) variabilities were estimated by standard deviations. Studied indices were the SBP-T variability ratio (dS/dT) and the DBP-T variability ratio (dD/dT). Standardized hazard ratios and odds ratios were determined using Cox regression and logistic regression, respectively., Results: Significant covariates adjusted hazard ratios for dS/dT and dD/dT that gradually increased across the following cohorts: entire sample, high-median age and high-median age and heart period: mean [95% confidence interval (95% CI)] 1.23 (1.03-1.47), 1.26 (1.05-1.52) and 1.55 (1.19-2.02), and 1.36 (1.07-1.72), 1.40 (1.09-1.80) and 1.57 (1.15-2.14), respectively. The corresponding odd ratios were similar in all cohorts: about 1.5 (1.2-2.0) and 1.3 (1.1-1.5). The adjusted indices size was greater in nonsurvivors (P =0.000001 and P = 0.003, respectively)., Conclusion: The 24-h BP to heart period variability ratios are powerful independent predictors of all-cause mortality, especially for elderly hypertensive patients with slow heart rate. The results support their interpretation as integrative indices of cardiovascular function and markers for cardiovascular dysregulation during low DBP states, with potential use in clinical practice.

Objectives: The slope of the linear relationship between systolic blood pressure (SBP) and diastolic blood pressure (DBP) determined from 24-h ambulatory measurements incorporates clinical and prognostic information and is believed to reflect arterial properties. We evaluated the feasibility of determining a similar relationship using the gravity-induced brachial blood pressure (BP) variation in response to vertical cuff displacement with reference to the heart., Methods: Nine consecutive BP readings were taken in the sitting position with arm postures stabilized by a mechanical support and with the cuff positioned at four different measured heights. The SBP-on-DBP slope (dS/dD) was estimated on the basis of the SBP and DBP variability ratio and expressed in terms of SD. The test was applied to 30 unselected volunteers (52% men, age 48±14)., Results: A mean cuff elevation of 32 cm was associated with a BP reduction of 21/19 mmHg (P<0.00001) without changes in the heart rate. The SBP-DBP correlation was 0.94±0.03 and the dS/dD was 1.10±0.03, with a determination error of 13±4% (mean±SE). Our results were similar to those of a previous study (n=37) having the same protocol, but in which the arm was self-supported. A case report that included 20 dS/dD determinations over 5 consecutive days and nights without height measurements demonstrated the repeatability of dS/dD, with a mean test duration of 13 min., Conclusion: The present study provides a novel, simple, and rapid method of attaining vascular-related information and its variation over time from BP measurements, with good accuracy that is applicable to both office and home. However, comparability with the ambulatory method, prognostic significance, and outcome variation over time remain to be evaluated.

Measures derived from the slope of the linear relationship between systolic and diastolic pressures obtained by 24-h ambulatory blood pressure (ABP) measurements incorporate clinical and prognostic information, and are believed to be vascular markers. Using post hoc analysis, we investigated potential changes of these 'slope-related measures' in three different studies conducted in hypertensive patients with before and after 24-h ABP measurements, and also evaluated the sensitivity of the results to the analysis method. Two interventional studies included 8-week device-guided breathing (DGB) exercised by 13 patients with uncontrolled blood pressure (BP), and a 6-month mineral potassium chloride-enriched diet administered to 20 elderly patients. One study was observational and involved winter-to-summer change experienced by 13 patients with controlled BP. Slope-related measures included systolic-on-diastolic slope and its equivalent 1-(diastolic-on-systolic slope) called Ambulatory Arterial Stiffness Index, and were determined using three different BP-averaging methods and two types of regression procedures. Results demonstrated sensitivity of slope-related measures to the analysis method, the most significant changes were found when the before and after 24-h ABP profiles included hourly averaged BP further averaged over the patient population, and slope-related measures were determined using symmetric (and not standard) regression. DGB was found to reduce significantly all these measures. The changes in the slope-related variables for individual patients correlated negatively with its baseline value and positively with the observed pulse pressure changes. In conclusion, the study provides evidence that DGB can affect positively vascular markers associated with cardiovascular risk, and suggests improved analysis methods for the determination of slope-related measures in interventional studies.

Objective: To challenge the widely accepted explanation of hydrostatic effect for blood pressure (BP) variation in response to arm elevation by testing the hypothesis that SBP and DBP measurements in the same individual vary differently with cuff height, and to evaluate the potential deviation of these measurements from the value predicted by the hydrostatic theory., Methods: Arm BP was measured in 37 individuals from the general population in the sitting position using a digital BP monitor. Measurements included nine consecutive BP readings at four cuff heights. The height dependence of BP was derived using reduced major axis regression., Results: The variation in systolic and diastolic pressure had a highly linear correlation with cuff height (r =  -0.90 and -0.93, respectively), with the systolic rate lower than the diastolic one by -0.13 ± 0.20 mmHg/cm (P = 0.0003) for all individuals. Systolic and diastolic rates were more negative in hypertensive individuals (P < 0.001 and P < 0.05, respectively). Significant deviation from the hydrostatic prediction of -0.77 mmHg/cm was observed in 30% of the individuals., Conclusion: This study assesses for the first time the inappropriateness of the hydrostatic theory as the sole explanation for the effect of gravity on BP. The phenomenon may be associated with mechanical properties of arteries in individuals known to have prognostic significance. The potential of the change in systolic and diastolic pressure with cuff height to be used as a novel type of clinical variable and vascular marker that can be determined by a simple test remains to be explored.

Routine practice of slow breathing is a major component of a variety of behavioral treatments of chronic conditions. The present paper addresses the challenge of using device-guided breathing (DGB) as a self-treatment tool in the home setting. The breathing patterns of hypertensive patients treated using DGB over 8 weeks in 15-min daily sessions were analyzed using minute-by-minute stored data. The relationship of breathing pattern with office blood pressure changes over the treatment period was evaluated. The treatment device operated to reduce breathing rate effortlessly while prolonging exhalation: patients were requested to synchronize breathing with the guiding tones, generated in response to the monitored breathing pattern. Breathing modification was highly individualized, repeatable over sessions, and unrelated to clinical or demographic characteristics; it also seemed to reflect behavioral aspects of device-patient interaction. Quality of breathing synchronization with the guiding tones and time spent in slow breathing displayed a relationship with the clinical outcomes. Different DGB methods are believed to share similar features. A possible physiological mechanism underlying the beneficial effect of slow and effortless breathing in chronic conditions is discussed in relation to restoring autonomic balance via lung reflexes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We explored the predictive ability of the blood pressure variability ratio (BPVR), defined as the ratio of 24-h ambulatory systolic blood pressure variability to diastolic variability, and evaluated its predictable relation with blood pressure and the Ambulatory Arterial Stiffness Index (AASI). A total of 3433 consecutive patients were followed up to 16 years for all-cause mortality. Blood pressure variability was expressed by the standard deviation. BPVR, which is the systolic-on-diastolic slope estimated by a known type of symmetric regression ('reduced major axis'), was compared with other expressions of this slope and with AASI using other regression procedures. Time-dependent Cox proportional hazard models, adjusted for demographics, 24-h mean blood pressure, 24-h pulse pressure and dipping were used to assess the association of BPVR and slope-related parameters with all-cause mortality. We found that Pearson's correlation between BPVR and the symmetric slope was 0.957, and between 1-1/BPVR (an AASI-equivalent expression) and the symmetric version of AASI was 0.973. BPVR was entirely independent of mean arterial pressure (r=0.013). Systolic and diastolic ambulatory blood pressure variability was not significantly associated with mortality. Over 16 years, BPVR predicted all-cause mortality [hazard ratio=1.21 (95% CI 1.05-1.40) per 1 s.d. increase]. In time-dependent models, increased BPVR was strongly associated with an 18-month mortality, weakly related to 7 years mortality, showing no effect thereafter. Thus, the ratio between 24-h systolic and diastolic blood pressure variability, readily available from ambulatory monitoring reports, is an easy-to-calculate systolic-on-diastolic slope. It is a blood pressure-independent measure believed to express an arterial property, with prognostic power similar to that of AASI.

The objective of the study was to evaluate the efficacy of device-guided breathing to lower blood pressure (BP) in hypertensive type II diabetic patients. A randomized controlled trial was carried out in four urban family practice clinics in Israel. Non-insulin-dependent diabetic, hypertensive patients with uncontrolled BP, receiving antihypertensive therapy or those non-medicated were enrolled. Baseline characteristics of the 66 patients who completed the study (33 intervention and 33 control) were: 62% men, age 62+/-8 years (mean+/-s.d.); body mass index 29+/-5 kg/m2; systolic BP 148+/-11 mm Hg and diastolic BP 81+/-9 mm Hg. The intervention group used a device (RESPeRATE), which interactively guides the user towards slow and regular breathing by synchronizing respiration voluntarily to musical tones for 15 min daily for an 8-week period. The control group continued with their regular treatment. BP was measured in the clinic at baseline, after 4 weeks and at 8 weeks. Medication was unchanged for 4 weeks prior to and during the study period. The main outcome measure was the office BP change from baseline to the end of the 8-week period. BP was reduced in the treatment group (mean+/-s.e.) systolic -10.0+/-1.8 mm Hg and diastolic -3.6+/-1.3 mm Hg (P<0.0001 and P<0.01), but not in the controls +1.6+/-2.1 and -1.0+/-1.4 mm Hg P>0.4 and P>0.4, respectively. Test for between group difference P<0.0001 and P=0.08. The subjects were highly compliant with the treatment, performing 75% of the requested exercise sessions. Greater BP reduction was observed with increased compliance with device usage (P=0.01 and P=0.001). It is concluded that self-treatment with device-guided breathing at home for 8 weeks by non-insulin-dependent diabetic patients was associated with a substantial reduction in office systolic BP.

Dependence of the ambulatory arterial stiffness index (AASI) on data scattering interferes with its potential clinical relevance. We assessed the correlates and all-cause mortality associations of a modified AASI (s-AASI). AASI was derived from the 24-h diastolic vs. systolic blood pressure linear regression line, whereas s-AASI was derived by symmetric regression (bisecting the line of diastolic vs systolic and systolic vs. diastolic). Of 2918 patients 55% were women; age was 56 +/- 16 years and body mass index was 27.3 +/- 4.5 kg/m(2). Average 24-h ambulatory blood pressure was 138 +/- 16/78 +/- 10 mm Hg. Applying the modified method for calculating AASI yielded a different measure: the negative correlation between AASI and blood pressure dipping (r = -0.304, P < 0.0001) was abolished (r = +0.223, P < 0.0001), s-AASI was more dependent on age (r = 0.266 vs. r = 0.089 for AASI), and prediction of all-cause mortality was enhanced; hazard ratio (95% confidence intervals) 1.17 (1.00-1.36) per 1 s.d. increase in s-AASI in the fully adjusted model as compared with 1.15 (0.97-1.36) for AASI.

Background: Regular slow breathing is known to improve autonomic cardiac regulation and reduce chemoreflex sensitivity in heart failure. We explored the acceptability and usefulness of a device for paced slow breathing at the home setting., Methods and Results: In this open pilot study, 24 patients with chronic heart failure (61% males, mean age, 64+/-9 years; New York Heart Association class, 2.81+/-0.01) were randomized to a control group receiving conventional treatment (n=12) or to a group receiving conventional treatment and device-guided paced breathing (n=12). Groups were comparable for age, therapies, and clinical characteristics. They were evaluated at baseline and again after 10 weeks by Doppler echocardiography, pulmonary function, cardiopulmonary stress test, and quality of life (Minnesota Quality of Life questionnaire). The treatment group was instructed to use the equipment for 18 minutes twice daily. The device is a computerized box connected to a belt-type respiration sensor and to headphones; it generates musical tones (based on the user's breathing rate and inspiration ratio), which guide the user to progressively and effortlessly slow his or her breathing rate <10 breaths/min. The treatment group showed high compliance to the device (90% of the prescribed sessions were completed). Blinded analysis of data demonstrated increased ejection fraction and decreased estimated pulmonary pressure in the echocardiograms of the treated group versus controls and favorable changes in New York Heart Association class, Ve/Vco(2), FEV(1), and a quality of life measure, as well (all P<0.05)., Conclusions: This pilot investigation demonstrates that device-guided paced breathing at home is feasible and results in an improvement in clinically relevant parameters for patients with heart failure and systolic dysfunction.

Objectives: Systolic blood pressure (SBP) and diastolic blood pressure (DBP) frequently display a linear relationship characterized by the systolic-versus-diastolic slope ('Slope') or the 'ambulatory arterial stiffness index' [AASI = 1 - (diastolic-versus-systolic Slope)] and the correlation coefficient r. We evaluated the effect of using symmetric regression on the AASI and its dependence on clinical characteristics using 24-h ambulatory monitoring., Methods: Ambulatory monitoring data of 140 patients (age 56 +/- 17 years, 45% men) were retrieved from a service database. Slope and the AASI were evaluated using symmetric regression procedures, and the AASI also by standard regression., Results: Correlation between SBP and DBP was r = 0.74 +/- 0.14 (r > 0.5 in 95% of patients). Low r-values (when SBP correlates poorly with DBP) were tightly linked with nondipping (P < 0.00001). Use of symmetric rather than standard regression eliminated the bias in slope-related parameters and unmasked their dependence on clinical characteristics. Both symmetric Slope and the AASI were independent of mean arterial pressure and r, increased with pulse pressure (P < 0.01 and P < 0.0001, respectively), with the greater effect of wider pulse pressure in older age (P < 0.005 for both). The symmetric slope was 1.29 +/- 0.28, showing bivariate dependence (r = 0.82) on age (exponential, with P < 0.00001) and pulse pressure dipping (P < 0.00001), increased for antihypertensive drug treatment (0.07 +/- 0.03, P < 0.05) and diabetes mellitus (0.18 +/- 0.06, P < 0.005)., Conclusions: Application of symmetrical regression provides a more valid estimate of the systolic-on-diastolic slope and the AASI, less influenced by goodness of fit and nocturnal dipping and more sensitive to age and disease states such as hypertension and diabetes, thus providing an improved index of arterial stiffening.

To study the effects of device-guided breathing on office systolic blood pressure (SBP), five centers randomized 149 untrained hypertensives (50% male, age 59+/-10 years, baseline blood pressure 150+/-9/86+/-9 mm Hg, 77% taking drug therapy). One half received a device to guide slow breathing; all received a home blood pressure monitor and only simple, written instructions. The changes in office SBP (adjusted for office-to-home difference in baseline SBP and accumulated time spent in slow breathing, guided and measured by the device) were significantly (p<0.001 for trend) correlated with accumulated time spent in slow breathing. Greater decreases in SBP (-15.0+/-1.8 vs. -7.3+/-1.9 mm Hg) were observed for those who spent more (vs. less) than 180 minutes over 8 weeks in slow breathing, as well as those who just monitored their blood pressure at home (-9.2+/-1.6 mm Hg). Thus, even without training, hypertensive patients who receive a device to guide slow breathing significantly lowered their office SBP if the total time spent in slow breathing over 8 weeks exceeded a "threshold" value of 180 minutes.

Background: Recent studies have demonstrated the antihypertensive effect of slow breathing exercises, guided interactively by a device, in patients with uncontrolled blood pressure (BP) without changing medication. This study examined the response to the same treatment protocol in resistant hypertensives., Methods: Seventeen resistant hypertensives exercised device-guided slow breathing for 8 weeks, 15 min daily, and self-monitored BP. Data stored in the devices were collected on a PC-based system. Clinical outcomes were office and home BP changes from baseline to end values., Results: Significant reductions in both office BP (-12.9/-6.9 mm Hg, P <.001 and home BP (-6.4/-2.6 mm Hg, P <.01/P <.05) without side effects with 82% responders and good compliance., Conclusions: Resistant hypertensives can benefit from and are compliant with self-treatment by device-guided slow breathing.

Objective: To examine the efficacy of a new device, which slows and regularises breathing, as a non-pharmacological treatment of hypertension and thus to evaluate the contribution of breathing modulation in the blood pressure (BP) reduction., Design and Setting: Randomised, double-blind controlled study, carried out in three urban family practice clinics in Israel., Patients: Sixty-five male and female hypertensives, either receiving antihypertensive drug therapy or unmedicated. Four patients dropped out at the beginning of the study., Intervention: Self treatment at home, 10 minutes daily for 8 consecutive weeks, using either the device (n = 32), which guides the user towards slow and regular breathing using musical sound patterns, or a Walkman, with which patients listened to quiet music (n = 29). Medication was unchanged 2 months prior to and during the study period., Main Outcome Measures: Systolic BP, diastolic BP and mean arterial pressure (MAP) changes from baseline., Results: BP reduction in the device group was significantly greater than a predetermined 'clinically meaningful threshold' of 10.0, 5.0 and 6.7 mm Hg for the systolic BP, diastolic BP and MAP respectively (P = 0.035, P = 0.0002 and P = 0.001). Treatment with the device reduced systolic BP, diastolic BP and MAP by 15.2, 10.0 and 11.7 mm Hg respectively, as compared to 11.3, 5.6 and 7.5 mm Hg (P = 0.14, P = 0.008, P = 0.03) with the Walkman. Six months after treatment had stopped, diastolic BP reduction in the device group remained greater than the 'threshold' (P < 0.02) and also greater than in the walkman group (P = 0.001)., Conclusions: The device was found to be efficacious in reducing high BP during 2 months of self-treatment by patients at home. Breathing pattern modification appears to be an important component in this reduction.

We hypothesise that routinely applied short sessions of slow and regular breathing can lower blood pressure (BP). Using a new technology BIM (Breathe with Interactive Music), hypertensive patients were guided towards slow and regular breathing. The present study evaluates the efficacy of the BIM in lowering BP. We studied 33 patients (23M/10F), aged 25-75 years, with uncontrolled BP. Patients were randomised into either active treatment with the BIM (n = 18) or a control treatment with a Walkman (n = 15). Treatment at home included either musically-guided breathing exercises with the BIM or listening to quiet music played by a Walkman for 10 min daily for 8 weeks. BP and heart rate were measured both at the clinic and at home with an Omron IC BP monitor. Clinic BP levels were measured at baseline, and after 4 and 8 weeks of treatment. Home BP measurements were taken daily, morning and evening, throughout the study. The two groups were matched by initial BP, age, gender, body mass index and medication status. The BP change at the clinic was -7.5/-4.0 mm Hg in the active treatment group, vs -2.9/-1.5 mm Hg in the control group (P = 0.001 for systolic BP). Analysis of home-measured data showed an average BP change of -5.0/-2.7 mm Hg in the active treatment group and -1.2/+0.9 mm Hg in the control group. Ten out of 18 (56%) were defined as responders in the active treatment group but only two out of 14 (14%) in the control group (P = 0.02). Thus, breathing exercise guided by the BIM device for 10 min daily is an effective non-pharmacological modality to reduce BP.

Slow breathing practiced routinely using an interactive device has demonstrated a sustained reduction in high blood pressure (BP). We reevaluated the BP response of hypertensives (n = 13) to this daily treatment for 8 weeks using 24-h ambulatory, home, and office BP measurements. A clinically significant BP reduction of similar magnitude was observed in all BP monitoring modalities during the daytime. Greater BP reductions were found for older patients and higher baseline BP. The results provide additional support for the efficacy of the device as an adjunctive lifestyle modification for treating hypertension.

The partial specific volume (V) and adiabatic compressibility (beta) of myoglobin have been shown to be reduced by small cosolvents such as glycerol (A. Priev, A. Almagor, S. Yedgar, B. Gavish, Biochemistry 35 (1996) 2061-2066). To elucidate the effect of the cosolvent size on these protein properties, in the present study we determined V and beta of myoglobin in solutions containing a homologous cosolvent series from sucrose to dextran--500 (M.W. 500,000). It was found that in addition to the expected effect of the cosolvent concentration, V and beta decrease with increasing cosolvent M.W. This suggests that structural properties of the cosolvent contribute to its effect on the protein interior.

Background: No information is available on the effects of vasoactive drugs on the microcirculation and on the correlation between their effects on the capillary and the systemic circulation., Purpose: To characterize the effects of nitroglycerin (NTG), nitroprusside (NP), and metaraminol infusion on microcirculatory variables using a noninvasive monitoring system., Methods: Increasing doses of NTG (5-40 microg/kg/min) and NP (5-20 microg/kg/min) were intravenously infused to eight rabbits. The microcirculatory parameters were monitored by a combined noninvasive system which included laser Doppler flowmetry (LDF), photoplethysmograph (PPG), and transcutaneous oxygen tension (tc-PO2). Mean blood pressure (MBP) was measured simultaneously., Results: At maximal rate of NTG infusion, the values of LDF, PPG, tc-PO2, and MBP were 72.2 +/- 23.9, 234.7 +/- 165.6, 103.5 +/- 37.4, and 82.7 +/- 7.4% of baseline, respectively. Following NP infusion the LDF, PPG, tc-PO2, and MBP reached 70.8 +/- 24.1, 190.5 +/- 68.9, 70.0 +/- 14.7, and 75.6 +/- 14.6% of baseline, respectively. The tissue oxygenation was significantly reduced during NP infusion compared to NTG (P < 0.05). The values gradually returned to the baseline 10 min after termination of NP infusion, but not after NTG. After injection of 1 mg metaraminol there was a steep decline in LDF, PPG, and tc-PO2 parallel to a significant increase in MBP. When metaraminol was administered after injection of 0.1 mg phenoxybenzamine, these changes were abolished., Conclusions: This noninvasive system effectively monitors microcirculatory hemodynamic changes induced by vasoactive drugs. NTG and NP cause similar changes in the systemic and microcirculatory bed, with the exception of peripheral oxygenation, which is reduced only during infusion of NP. The changes in the microcirculatory bed following metaraminol and phenoxybenzamine imply that their effects on the arterioles are mediated by adrenergic alpha-receptors.

The addition of hydrogen-bonded cosolvents to aqueous solutions of proteins is known to modify both thermodynamic and dynamic properties of the proteins in a variety of ways. Previous studies suggest that glycerol reduces the free volume and compressibility of proteins. However, there is no directly measured evidence for that. We have measured the apparent specific volume (V) and adiabatic compressibility (K) of a number of proteins, sugars, and amino acids in water and in 30% glycerol at pH 7.4 and 30 degrees C. The values of V and K in water and their changes induced by glycerol were extrapolated to the limit of infinite solute size. The main results were the following: (a) glycerol decreases V and K of proteins, but increases it for amino acids; (b) the V and K values of the protein interior in water were found to be 0.784 +/- 0.026 mL/g and (12.8 +/- 2.5) x 10(-6) mL/g x atm, where the glycerol reduces these values by 8 and 32%, respectively; (c) the coefficient of adiabatic compressibility of the structural component of proteins affected by the glycerol is estimated to be (50 +/- 10) x 10(-6) atm(-1), which is comparable to that of water. We propose that the glycerol induces a release of the so-called "lubricant" water, which maintains conformational flexibility by keeping apart neighboring segments of the polypeptide chain. This is expected to lead to the collapsing of the voids containing the water as well as to increase intramolecular bonding, which explains the observed effect.

The purpose of the study was to examine the ability of a system combining laser Doppler flowmetry (LDF), photoplethysmograph (PPG), and transcutaneous oxygen tension (tc-PO2) to follow changes in the microcirculation during hemorrhage and following blood or saline return, and to test the hypothesis that such changes precede and might predict changes in the systemic blood pressure. Measurements were performed on the skin of anesthetized rabbits (n = 10) during mild (0-8%), moderate (9-24%), and severe (25-30% of blood volume) hemorrhage, and following complete volume restitution by blood or saline. We found the following: 1) hemorrhage caused typical changes in the LDF, PPG, and tc-PO2 signals that could be formulated by mathematical models, 2) these signals identified blood as being more efficient than saline for volume restitution following hemorrhage, and 3) microcirculatory changes precede and might predict systemic hemodynamic events.

The morphology of red blood cell (RBC) aggregates was studied by direct visualization of RBC aggregation at different flow conditions in a computerized image analyzer. The aggregate morphology is expressed by an Aggregate Shape Parameter (ASP), defined as the ratio of the aggregate projected area to its square perimeter. Aggregation was induced by either dextran-70 (m.w. 70,000) or dextran-500 (m.w. 500,000), and compared to that in plasma. It was found that the aggregate morphology is a characteristic of the aggregating agent--in dextran-500, the RBC form rouleau aggregates as in plasma, while in dextran-70, they form clusters. In each system, while maintaining the overall typical morphology, the ASP decreases (i.e., the aggregate becomes longer) as the aggregate size is increased. The distribution of the ASP as a function of the aggregate size remains unchanged when the aggregate size is changed by modulation of the dextran concentration or the shear stress. Stretching of a rouleau aggregate by application of shear stress is reflected by a corresponding change in the ASP. It is suggested that the ASP is a characteristic of intercellular interactions. A theoretical model is proposed for evaluation of the deviation of aggregate shape from that of rouleau structure.

Laser photodissociation of respiratory proteins is followed by fast geminate recombination competing with escape of the oxygen molecule into the solvent. The escape rate from myoglobin or hemerythrin has been shown previously to exhibit a reciprocal power-law dependence on viscosity. We have reinvestigated oxygen escape from hemerythrin using a number of viscous cosolvents of varying molecular weight, from glycerol to dextrans up to 500 kDa. In isoviscous solutions, the strong viscosity dependence observed with small cosolvents is progressively reduced upon increasing the cosolvent's molecular weight and disappears at molecular weights greater than about 100 kDa. Thus, viscosity is not a suitable independent parameter to describe the data. The power of the viscosity dependence of the rate coefficient is shown here to be a function of the cosolvent's molecular weight, suggesting that local protein-solvent interactions rather than bulky viscosity are affecting protein dynamics.

The effect of hydrostatic pressure of up to 15 bars on the aggregability of rat and human red blood cells (RBC), i.e., their capability to form aggregates, was studied using computerized image analysis. The aggregate size distribution was determined under ambient pressure, following application of hydrostatic pressure for various durations up to 2 h. It was found that RBC aggregability markedly increases, up to three-fold, as the pressure which had been applied was increased. Accordingly, higher shear stress is required for dispersing the aggregates of pressure-treated RBC than those of untreated cells. The median size of human RBC aggregates was about three times higher than that of rat RBC, and this ratio was maintained following pressure treatment. RBC aggregability is a major determinant in blood flow, especially in the microcirculation. Pressure at the levels used in this study occurs in physiological states such as hyperbaric treatment or diving. The enhanced aggregability induced by application of such pressure implies that blood flow in microvessels might be altered under conditions associated with elevated hydrostatic pressure.

Protein-ligand binding and enzyme activity have been shown to be regulated by solvent viscosity, induced by the addition of viscous cosolvents. This was indirectly interpreted as an effect on protein dynamics. However, viscous cosolvents might affect dynamic, e.g., viscosity, as well as thermodynamic properties of the solution, e.g., activity of solution components. This work was undertaken to examine the effect of viscous cosolvent on the structural dynamics of proteins and its correlation with dynamic and thermodynamic solution properties. For this purpose we studied the effect of viscous cosolvent on the specific ultrasonic absorption, delta mu, of bovine serum albumin, at pH = 7.0 and at 21 degrees C, and frequency range of 3-4 MHz. Ultrasonic absorption (UA) directly probes protein dynamics related to energy dissipation processes. It was found that the addition of sucrose, glycerol, or ethylene glycol increased the BSA delta mu. This increase correlates well with the solvent viscosity, but not with the cosolvent mass concentration, activity of the solvent components, dielectric constant, or the hydration of charged groups. On the grounds of these results and previously reported findings, as well as theoretical considerations, we propose the following mechanism for the solvent viscosity effect on the protein structural fluctuations, reflected in the UA: increased solvent viscosity alters the frequency spectrum of the polypeptide chain movements; attenuating the fast (small amplitude) movements, and enhancing the slow (large amplitude) ones. This modulates the interaction strength between the polypeptide and water species that "lubricates" the chain's movements, leading to larger protein-volume fluctuation and higher ultrasonic absorption. This study demonstrates that solvent viscosity is a regulator of protein structural fluctuations.

Solvent viscosity is known to play an important role in the kinetics of biochemical reactions, and has been suggested to modulate the dynamic structure of proteins. The effect of viscous cosolvents, of various molecular sizes, on the apparent ultrasonic absorption of bovine serum albumin in solution, at 37 degrees, has been measured in attempt to investigate the following phenomena: 1) The predicted modulating effect of viscous cosolvents on the "internal friction" of proteins, and 2) Possible differences between the microscopic and macroscopic pictures of the solvent viscosity concerning the proposed effect. We have found that A) The absorption of ultrasound (3-17 MHz) by the protein increases with increasing the cosolvent concentration. B) That increase correlates with the solvent viscosity for small cosolvent molecules, but not with macromolecular cosolvents, and C) Dextran solutions with the same concentration by weight, reveal similar ultrasonic absorption, in spite of large differences in their viscosity. A possible explanation is discussed.

The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.

The effects of pH changes on the miniature endplate current (mepc) and on endplate current fluctuations (acetylcholine [ACh] noise) were examined at the neuromuscular junction in vitro in two species of frogs. In Rana pipiens the relationship between the decay time constant of the mepc (tau') and pH had a symmetrical bell shape; the value of tau' being largest at pH 7 and decreasing at more acid or more alkaline pH. In acid pH the mepc amplitude (A) decreased relative to its value at pH 7, and in alkaline pH A increased. In Rana ridibunda a narrower and asymmetric bell-shaped dependence of tau' on pH, having a maximum of pH 5.5, was found. The mepc amplitude was again reduced in acid pH but had a peak at pH 5.5. Also, its value at pH 9 was larger than at pH 7. These results were obtained with a number of different buffers and were not found to be sensitive to the nature of the buffer chosen. By performing ACh-noise analysis we found that in Rana pipiens at acid pH (5.5-5.0), the single channel conductance (gamma) and the single channel open time (tau) were significantly reduced relative to their value at pH 7. However, in Rana ridibunda at acid pH (5.4) gamma was unchanged and tau was markedly increased relative to their values at pH 7. The results can be explained quantitatively by electrostatic interaction between two fixed and titratable ionic groups and a mobile charge in the receptor molecule. The model fits the data for groups having pKs approximately 4.8 and approximately 9.8 for Rana pipiens and approximately 4.6 and approximately 6.3 for Rana ridibunda. The groups can be tentatively identified as amino acid residues; glutamic or aspartic and lysine or tyrosine for Rana pipiens; glutamic or aspartic and histidine for Rana ridibunda. The difference in the fitted values of the other model parameters for these two species can be attributed to differences in the spatial configuration of the charged groups.

Time course measurements of the action of muscarinic antagonists were performed in the spontaneously beating carp atrium. Several high affinity drugs, which embody the quinuclidine structure were examined. The structural flexibility of these molecules was reflected in the dissociation of the drugs from the muscarinic receptor. The dissociation of rigid drugs was very much prolonged as compared to flexible drugs of the same affinity.