Optical bio-markers have been used extensively for intracellular imaging with high spatial and temporal resolution. Extending the modality of these probes is a key driver in cell biology. In recent ...years, the NV centre in nanodiamond has emerged as a promising candidate for bio-imaging and bio-sensing with low cytotoxicity and stable photoluminescence. Here we study the electrophysiological effects of this quantum probe in primary cortical neurons. Multi-electrode array (MEA) recordings across five replicate studies showed no statistically significant difference in 25 network parameters when nanodiamonds are added at varying concentrations over various time periods 12-36 hr. The physiological validation motivates the second part of the study which demonstrates how the quantum properties of these biomarkers can be used to report intracellular information beyond their location and movement. Using the optically detected magnetic resonance from the nitrogen vacancy defects within the nanodiamonds we demonstrate enhanced signal-to-noise imaging and temperature mapping from thousands of nanodiamond probes simultaneously. This work establishes nanodiamonds as viable multi-functional intraneuronal sensors with nanoscale resolution, that may ultimately be used to detect magnetic and electrical activity at the membrane level in excitable cellular systems.
Hypotheses regarding the pathogenesis of volume-dependent hypertension have invoked an endogenous sodium pump inhibitor or digitalis-like factor (DLF) to link altered sodium homeostasis to the rise ...in blood pressure. Our goal was to develop a clinical protocol that achieved predictable, sustained volume expansion, with the premise that renal failure patients on peritoneal dialysis would increase intravascular volume, gain weight, and raise blood pressure (BP) in relation to measured increases in DLF. In a 5-day protocol, dialysis was kept constant but dietary NaCl and fluids were modified in 7 patients. DLF was measured as inhibition of Na,KATPase. Likewise, the first 2 L of daily peritoneal dialysate (PD) was processed on HPLC and the eluate analyzed for DLF. The group achieved significant weight gain (WT) by day 3 (delta WT = 4.1 +/- 1.2 kg, P < .05). Likewise, mean arterial pressure (MAP) and plasma DLF activity increased significantly. All variables were highly correlated (DLF v WT: R = 0.88, P = .004; MAP v DLF: R = 0.82, P = .01; MAP v WT: R = 0.90, P = .003). Although a number of HPLC fractions contained agents that interacted with the assay, only one PD HPLC fraction (at 19.5 min) contained DLF activity that correlated with changes in MAP (R = 0.60, P = .002), and body weight (R = 0.67, P = .0003). We conclude that candidate DLF responds to sustained volume expansion and the relationship suggests that it could influence blood pressure. Moreover, the application of stringent criteria to the confusing array of factors in plasma that may affect assays for DLF appears to reduce the field dramatically, to a single candidate in this setting.
The possibility that a circulating sodium pump inhibitor contributes to the pathogenesis of volume-dependent hypertension via an action on vascular smooth muscle (VSM) is supported by multiple lines ...of investigation, but remains controversial. We had two goals in this study. The first was to compare the pattern of contractile response of rabbit aorta induced by two candidates, ouabain and a labile sodium pump inhibitor that we have identified in the peritoneal dialysate of volume-expanded hypertensive patients with chronic renal failure. Our second goal was to examine the ability of Digibind, a Fab fragment of antisera directed against digoxin, to reverse VSM contraction induced by both agents. Ouabain induced a concentration-dependent contraction, which was delayed in onset, was gradual, and reached a stable plateau after many hours. The labile sodium pump inhibitor induced a qualitatively similar series of responses. Digibind rapidly reversed the contractile responses to both sodium pump inhibitors, with a rate of relaxation that matched that induced by physical removal of the pump inhibitor from the bath. For ouabain, the Digibind:ouabain stoichiometry was highly predictable. When Digibind was present in a molar concentration equivalent to that of ouabain, or less, it had no effect. When the Digibind concentration was twice that of ouabain, complete relaxation occurred. Although the concentration:VSM response relationship for ouabain was steep, the concentration:effect interaction with Digibind was even more steep. The molar concentration of Digibind required to reverse the effects of the labile endogenous inhibitor from peritoneal dialysate was consistently lower than that for ouabain, which is compatible with either greater potency of the labile factor in VSM or greater affinity for Digibind. These findings are compatible with a role for one or more endogenous sodium pump inhibitors as the determinant of vascular smooth muscle tone in the volume-sensitive hypertension of renal disease.
A volume-sensitive inhibitor of Na,KATPase, termed the digitalis-like factor (DLF), is postulated to participate in hypertension. To test this hypothesis, end-stage renal failure patients on ...peritoneal dialysis were placed on a clinical protocol that brought about a gradual, sustained volume expansion. This was accompanied by significant increases in body weight (4.1 +/- 1.2 kg, p < 0.05), mean arterial pressure (18.2 +/- 6.4 mm Hg, p < 0.05), and serum DLF activity (4.7 +/- 1.9% inhibition, p < 0.05). Processing these patients' daily dialysates by ultrafiltration and high-performance liquid chromatography allowed for the identification of a single elution fraction having volume-sensitive Na,KATPase inhibitory activity. This factor in turn was correlated with serum DLF activity (R = 0.60, p = 0.002), weight gain (R = 0.67, p = 0.0003), and mean arterial pressure (R = 0.59, p = 0.003). This factor was readily distinguished from ouabain and digoxin but was similar to the DLF isolated from amniotic fluid. These results suggest that volume expansion in renal failure patients on peritoneal dialysis gives rise to a unique volume-sensitive DLF that may contribute to these patients' increase in blood pressure.
Increased levels of a circulating digoxin-like factor (DLF) occur in a number of physiologic states in which sodium homeostasis is altered, and may contribute to the pathogenesis of hypertension. We ...exploited the different affinities for DLF of seven antisera directed at digoxin to develop an immunochemical profile, and then employed this index to address two questions: does the same DLF species exist in several conditions associated with increased DLF levels, including pregnancy, renal failure, hepatic failure, and neonatal cord blood? Will this approach prove useful in assessing candidates proposed to be DLF? An identical profile was identified in serum from pregnant women and patients with renal or hepatic failure, and a highly significant correlation existed between DLF levels measured with antisera of high and intermediate affinity in 42 subjects with increased levels (r = 0.93; P less than .001). In patients with renal failure, when endogenous DLF levels were too low to assess the profile, concentration of the serum resulted in measurable DLF levels that had an identical profile. The profile was somewhat altered in umbilical cord blood, perhaps reflecting an influence of increased steroid hormone levels. Among agents suggested as candidates for DLF, neither lysophosphatidylcholine nor ouabain showed a profile resembling DLF. Progesterone, 17-OH-progesterone, and bufalin, on the other hand, did show substantial similarity, perhaps providing a clue to the structure of DLF. The normal plasma levels of progesterone and 17-OH-progesterone are 100- to 1000-fold too low to be candidates for DLF and bufalin was sufficiently dissimilar not to be a candidate. DLF in at least three different patient populations probably represents identical chemical species.
Epidemiological studies in China suggest that dietary zinc deficiency and environmental exposure to N-nitrosamine carcinogens, such as N-nitrosomethylbenzylamine (NMBA), are among the factors ...associated with an increased incidence of esophageal carcinoma in humans. NMBA belongs to a class of nitrosamines which require activation by the cytochrome P-450-dependent mixed-function oxidases in order to be mutagenic. Rats maintained on a zinc-deficient diet exhibited an increased incidence of NMBA-induced esophageal carcinoma when compared to rats on a control diet. The increased tumor formation was associated with an alteration of the microsomal metabolism of NMBA. Weanling male Sprague-Dawley rats were raised on egg protein diets containing 2.3 ppm zinc (low zinc) or 50 ppm zinc (control zinc). Analysis of tissues revealed a rapid decline in the levels of zinc in serum and esophagi of the animals fed the low-zinc diet. Gastric and hepatic zinc content did not differ significantly between the animals fed the low-zinc diet and the animals fed the control zinc diet, even after 6 weeks. Microsomes were prepared from esophageal mucosa, livers, and forestomachs from weanling animals fed these diets for 3 weeks. The rate of formation of benzaldehyde from NMBA by esophageal mucosal microsomes prepared from the rats fed the low-zinc diet was nearly 10-fold higher than that of the rats fed the control zinc diet 0.230 +/- 0.047 (S.E.) versus 0.024 +/- 0.008 nmol/min/mg microsomal protein; p less than 0.001. The rate of benzaldehyde formation by hepatic microsomes was 0.062 +/- 0.005 nmol/min/mg microsomal protein in the rats fed the low-zinc diet and 0.042 +/- 0.002 nmol/min/mg microsomal protein in the rats fed the control zinc diet (p less than 0.01). The rate of benzaldehyde formation by forestomach microsomes was not detectable in tissue from rats on either diet. This increased rate of NMBA metabolism by esophageal mucosal microsomes from the zinc-deficient rats may explain the increased incidence of esophageal carcinoma in these animals.
Smooth muscle responses to Na+ pump inhibition are thought to reflect two elements: a neurogenic contribution, involving catecholamine release from nerve terminals, and a myogenic response, ...attributed to relations between pump activity, Na+i, and Ca2+i. In the present study, we describe the time course and magnitude of cell Na+ changes, assessed by two methods, atomic absorption and nuclear magnetic resonance spectroscopy during the myogenic contractile response of rabbit aorta strips to ouabain. A threshold concentration of 3 x 10(-7) mol/L induced a gradual rise in Na+i. Both methods showed an essentially identical monotonic rise over 4 to 8 hours from a baseline level of 8 to 10 mmol/L water to a peak, which was approximately fivefold higher. The neurogenic (rapid) and myogenic (delayed and gradual) contractile responses were temporally distinct. Ouabain at 10(-7) mol/L, a concentration 10- to 100-fold lower than the threshold for catecholamine-dependent rapid-onset responses, induced only a delayed and gradual contractile response, which reached a maximum at 6 to 8 hours. With 10(-6) mol/L ouabain, the delayed response of 1.6 +/- 0.2 g peaked at 7.3 +/- 1.1 hours and was sustained for 16 hours. The time course was similar to that for change in Na+ but somewhat later. Ouabain at 10(-5) and 10(-4) mol/L induced a delayed response that was identical in magnitude but also induced an early rapid contractile response, which was prevented by reserpine or phentolamine pretreatment. These agents did not influence the delayed response.
We have reported that 50% of subjects with normal renin essential hypertension have both delayed suppression of the renin-angiotensin-aldosterone axis following sodium infusion and a delayed rate of ...excretion of an acute salt load. In another study we have also described a subset of patients with essential hypertension (called nonmodulators) who have several abnormalities, including a pressor response to salt loading. To evaluate whether the abnormalities described in these different groups of patients actually occur in the same patient, we assessed the renin-angiotensin-aldosterone axis response to short-term saline loading in 38 hypertensive patients. Their ability to modulate was determined by their renal vascular response to infused angiotensin II on a high salt diet (200 mEq Na). In response to a 3-hour infusion of saline, 75 mEq/hr, the reduction in plasma renin activity at both 60 and 120 minutes was significantly greater (p less than 0.008) in patients with normal modulation than in the nonmodulators. Plasma aldosterone levels were also significantly lower (p less than 0.001) in those with intact modulation. Thus, nonmodulating essential hypertensive patients have abnormalities in several systems that influence sodium homeostasis, including altered adrenal and renal vascular response to angiotensin II, altered renal blood flow response to salt loading, and a delayed suppression of the renin-angiotensin-aldosterone system with short-term saline infusion.
Alveolar epithelial beta-adrenergic receptor (betaAR) activation accelerates active Na+ transport in lung epithelial cells in vitro and speeds alveolar edema resolution in human lung tissue and ...normal and injured animal lungs. Whether these receptors are essential for alveolar fluid clearance (AFC) or if other mechanisms are sufficient to regulate active transport is unknown. In this study, we report that mice with no beta1- or beta2-adrenergic receptors (beta1AR-/-/beta2AR-/-) have reduced distal lung Na,K-ATPase function and diminished basal and amiloride-sensitive AFC. Total lung water content in these animals was not different from wild-type controls, suggesting that betaAR signaling may not be required for alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with beta1- but not beta2-adrenergic receptors to beta1AR-/-/beta2AR-/- mice indicates that the beta2AR mediates the bulk of beta-adrenergic-sensitive alveolar active Na+ transport. To test the necessity of betaAR signaling in acute lung injury, beta1AR-/-/beta2AR-/-, beta1AR+/+/beta2AR-/-, and beta1AR+/+/beta2AR+/+ mice were exposed to 100% oxygen for up to 204 hours. beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice had more lung water and worse survival from this form of acute lung injury than wild-type controls. Adenoviral-mediated rescue of beta2-adrenergic receptor (beta2AR) function into the alveolar epithelium of beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice normalized distal lung beta2AR function, alveolar epithelial active Na+ transport, and survival from hyperoxia. These findings indicate that betaAR signaling may not be necessary for basal AFC, and that beta2AR is essential for the adaptive physiological response needed to clear excess fluid from the alveolar airspace of normal and injured lungs.
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