Tonic and stimulus-evoked nitric oxide production in the mouse olfactory bulb.

Nitric oxide (NO) has been long assumed to play a key role in mammalian olfaction. This was based largely on circumstantial evidence, i.e. prominent staining for nitric oxide synthase (NOS) and cyclic guanosine 3',5'-cyclic monophosphate (cGMP) or soluble guanylyl cyclase, an effector enzyme activated by NO, in local interneurons of the olfactory bulb. Here we employ innovative custom-fabricated NO micro-sensors to obtain the first direct, time-resolved measurements of NO signaling in the olfactory bulb. In 400 microm thick mouse olfactory bulb slices, we detected a steady average basal level of 87 nM NO in the extracellular space of mitral or granule cell layers. This NO 'tone' was sensitive to NOS substrate manipulation (200 microM L-arginine, 2 mM N(G)-nitro-L-arginine methyl ester) and Mg(2+) modulation of N-methyl-D-aspartate (NMDA) receptor conductance. Electrical stimulation of olfactory nerve fibers evoked transient (peak at 10 s) increments in NO levels 90-100 nM above baseline. In the anesthetized mouse, NO micro-sensors inserted into the granule cell layer detected NO transients averaging 55 nM in amplitude and peaking at 3.4 s after onset of a 5 s odorant stimulation. These findings suggest dual roles for NO signaling in the olfactory bulb: tonic inhibitory control of principal neurons, and regulation of circuit dynamics during odor information processing.

Heterogeneous response of microvascular endothelial cells to shear stress.

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.

Heterogeneous cytoplasmic calcium response in microvascular endothelial cells.

We investigated changes in calcium concentration in response to the administration of ATP and the onset of shear stress with cultured rat adrenomedulary endothelial cells (RAMECs, microvascular). A substantial heterogeneity in time and space in the calcium response was observed. The onset of shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells The application of uniform exogenous ATP produced similar heterogeneous calcium transients. The size of the responding groups was dependent on ATP concentration. The propagation of calcium waves induced by either ATP or shear stress challenge was significantly suppressed by suramin, a non-specific purinergic receptor blocker. We investigated some of the mechanisms leading to the heterogeneity, and the results indicated that the main source of variation is the heterogeneous distribution of purinergic receptor. The application of ATP or shear stress stimulates cells to release ATP causing an increase of [Ca2+]ivia purinergic receptor in the cells that have high sensitivity. Subsequently, additional ATP is released and the elevation of ATP concentration in the vicinity of the initially responding cells mediates the calcium propagation. These data suggest a mechanism by which ATP acts as an autocrine and paracrine mediator to integrate individual cell responses that result in coordination of vascular functions in situ.

Can we model nitric oxide biotransport? A survey of mathematical models for a simple diatomic molecule with surprisingly complex biological activities.

Nitric oxide (NO) is a remarkable free radical gas whose presence in biological systems and whose astonishing breadth of physiological and pathophysiological activities have only recently been recognized. Mathematical models for NO biotransport, just beginning to emerge in the literature, are examined in this review. Some puzzling and paradoxical properties of NO may be understood by modeling proposed mechanisms with known parameters. For example, it is not obvious how NO can survive strong scavenging by hemoglobin and still be a potent vasodilator. Recent models do not completely explain how tissue NO can reach effective levels in the vascular wall, and they point toward mechanisms that need further investigation. Models help to make sense of extremely low partial pressures of NO exhaled from the lung and may provide diagnostic information. The role of NO as a gaseous neurotransmitter is also being understood through modeling. Studies on the effects of NO on O2 transport and metabolism, also reviewed, suggest that previous mathematical models of transport of O2 to tissue need to be revised, taking the biological activity of NO into account.

Temporal dynamics of the partial pressure of brain tissue oxygen during functional forepaw stimulation in rats.

The partial pressure of tissue oxygen (pO2) was measured in rat somatosensory cortex during periodic electrical forepaw stimulation of either 1 min or 4 s in duration, and correlated with simultaneous laser Doppler flowmetry. For both stimulus durations, a transient decrease in tissue pO2 preceded blood flow changes, followed by a peak in blood flow and an overshoot in tissue pO2. With protracted stimulation, tissue pO2 remained only slightly above pre-stimulus baseline, while blood flow was maintained at a reduced plateau phase. A sustained post-stimulus undershoot in tissue pO2 was observed only for the 1 min stimulus. These findings suggest a complex dynamic relationship between oxygen utilization and blood flow.

Regulation of oxygen sensing in peripheral arterial chemoreceptors.

The carotid bodies are a small pair of highly vascularized and well perfused organs located at each carotid artery bifurcation, strategically situated to sense oxygen in arterial blood as it leaves the heart. Carotid body glomus cells are identified as the primary oxygen sensors, which respond to changes in blood P(O(2)) within milliseconds. Acute hypoxia causes a rapid increase in carotid sinus nerve (CSN) activity, providing afferent signals to the respiratory center in the brainstem. Glomus cells secrete numerous neurotransmitters that modulate CSN firing rates. This review will discuss major hypotheses that have emerged regarding acute oxygen sensing by glomus cells. In contrast, chronic responses to hypoxia are much slower, involving cytosolic reactions that take place over several minutes and nuclear reactions which occur over several hours. Converging concepts from different areas of research in oxygen sensing cells and tissues (including the carotid body) have been combined to describe molecular and biochemical changes that take place in the carotid body with chronic hypoxia. These include oxygen dependent proteolytic processes in the cytosol and gene transcription in the nucleus. In addition, cellular and nuclear responses to chronic hypoxia will be discussed.

Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability.

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [l-N(6)-(1-iminoethyl) lysine, l-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) > or = WT with l-NIL or iNOS(-/-) > eNOS(-/-) > or = eNOS(-/-) with l-NIL. The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and iNOS(-/-) mice but not in eNOS(-/-) mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.

Dopamine, sensory discharge, and stimulus interaction with CO2 and O2 in cat carotid body.

It is hypothesized that carotid body chemosensory activity is coupled to neurosecretion. The purpose of this study was to examine whether there was a correspondence between carotid body tissue dopamine (DA) levels and neuronal discharge (ND) measured from the carotid sinus nerve of perfused cat carotid bodies and to characterize interaction between CO2 and O2 in these responses. ND and tissue DA were measured after changing from normoxic, normocapnic control bicarbonate buffer (PO2 >120 Torr, PCO2 25-30 Torr, pH approximately 7.4) to normoxic hypercapnia (PCO2 55-57 Torr, pH 7.1-7.2) or to hypoxic solutions (PO2 30-35 Torr) with normocapnia (PCO2 25-30 Torr, pH approximately 7.4) or hypocapnia (PCO2 10-15 Torr, pH 7.6-7.8). Similar temporal changes for ND and tissue DA were found for all of the stimuli, although there was a much different proportional relationship for normoxic hypercapnia. Both ND and DA increased above baseline values during flow interruption and normocapnic hypoxia, and both decreased below baseline values during hypoxic hypocapnia. In contrast, normoxic hypercapnia caused an initial increase in ND, from a baseline of 175 +/- 12 (SE) to a peak of 593 +/- 20 impulses/s within 4.6 +/- 0.9 s, followed by adaptation, whereas ND declined to 423 +/- 20 impulses/s after 1 min. Tissue DA initially increased from a baseline of 17.9 +/- 1.2 microM to a peak of 23.2 +/- 1.2 microM within 3.0 +/- 0.7 s, then declined to 2.6 +/- 1.0 microM. The substantial decrease in tissue DA during normoxic hypercapnia was not consistent with the parallel changes in DA with ND that were observed for hypoxic stimuli.

In vivo tissue pO2 measurements in hamster skinfold by recessed pO2 microelectrodes and phosphorescence quenching are in agreement.

OBJECTIVE: Phosphorescence quenching has been used successfully to optically measure in vivo blood pO2 in the microvasculature. Optical measurements have also been made in some tissues, but it is not clear whether these results accurately reflect tissue pO2. METHODS: Recessed pO2 microelectrodes and the phosphorescence quenching technique were used simultaneously to measure in vivo tissue pO2 in hamster skinfold. The optical window for phosphorescence quenching was focused around the tips of microelectrodes that were positioned in tissue regions at least 100 microns from large microvessels. RESULTS: Mean tissue pO2 measured by recessed pO2 microelectrodes was 18.4 +/- 1.7 (SE) Torr, and mean tissue pO2 determined from the time course of phosphorescence decay was 18.8 +/- 2.0 Torr (no significant difference). The two tissue pO2 measurements agreed over a wide range, from 2 to 46 Torr (r = 0.93, 39 paired measurements from six sites in 3 animals). There was no systematic change in the microelectrode tissue pO2 during the period of light excitation used for the optical method. CONCLUSIONS: Under the conditions of our study, sufficient amounts of porphyrin dye leaked from the vasculature and diffused into tissue, allowing accurate measurements of tissue pO2 by the phosphorescence quenching technique. Furthermore, the optical method did not deplete significant amounts of O2 from tissue during light excitation.

Inhibition of dopamine release with simultaneous chemosensory excitation by hypercapnia with and without [Ca2+]0 in the cat carotid body.

The hypothesis that dopamine (DA) overflow corresponds to carotid sinus nerve (CSN) discharge during hypercapnia and is dependent on [Ca2+]0 was tested. We simultaneously measured the time course of DA overflow and CSN discharge of the cat carotid body, perfused/superfused in vitro at 37 degrees C at decreasing [Ca2+]0, during transition from normocapnia (PCO2 approximately 30-35 Torr) to hypercapnia (PCO2 approximately 60-65 Torr). In the presence of normal [Ca2+]0, hypercapnia instantaneously increased nerve discharge to peak levels followed by a decrease to steady states which were above the basal rate of activity. CSN discharge rate did not differ at decreasing [Ca2+]0 between 2.2 and 1.0 mM, and it began to decline at 0.1 mM [Ca2+]0, culminating to zero level in most cases, at zero [Ca2+]0. DA overflow increased slightly during hypercapnic peak CSN activity. Thereafter it declined to steady state levels below those of normocapnic conditions. Decreases in steady state DA levels were significantly less at 0 mM [Ca2+]0 compared to the higher calcium concentrations (0.1, 1.0 and 2.2 mM). Overall, steady state CSN activity and DA overflow were inversely related. Thus, DA release cannot have excitatory implications for carotid chemoreceptors during hypercapnia in the cat.

Microvascular and tissue oxygen gradients in the rat mesentery.

One of the most important functions of the blood circulation is O2 delivery to the tissue. This process occurs primarily in microvessels that also regulate blood flow and are the site of many metabolic processes that require O2. We measured the intraluminal and perivascular pO2 in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O2 diffuses out of microvessels from the blood. The rate of O2 efflux and the O2 gradients found in the immediate vicinity of arterioles indicate the presence of a large O2 sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O2 from the arterioles in this vascular bed primarily is caused by O2 consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to parenchymal tissue in the rat mesentery suggests that in addition to serving as a conduit for the delivery of O2 the microvasculature has other functions that require a significant amount of O2.

Suppression of glomus cell K+ conductance by 4-aminopyridine is not related to [Ca2+]i, dopamine release and chemosensory discharge from carotid body.

The hypothesis that suppression of O2-sensitive K+ current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine (4-AP), a known suppressant of K+ current, on intracellular [Ca2+]i, dopamine secretion and chemosensory discharge in cat carotid body (CB). In vitro experiments were performed with superfused-perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP (0.2, 1.0 and 2.0 mM in CO2-HCO3- buffer) and recording [Ca2+]i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in [Ca2+]i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered [Ca2+]i by 4-AP are not consistent with the implied meaning of the suppressant effect on K+ current of glomus cells.

Vasomotion and spontaneous low-frequency oscillations in blood flow and nitric oxide in cat optic nerve head.

The purpose of this study was to determine whether spontaneous oscillations in blood flow (relative red blood cell flux) measured by laser Doppler flowmetry (LDF) in the cat optic nerve head were related to fluctuations in nitric oxide (NO) measured with electrochemical sensors (n = 16 cats). Power spectral densities for the magnitude and frequency of LDF and NO fluctuations were determined by discrete Fourier transform analysis. Complex behavior was found for both LDF and NO oscillations with broad spectra containing peaks at multiple frequencies. Most of the power was in the low-frequency range (<10 cycles/min). Spectra were also obtained after administering NO synthase inhibitors (l-nitroarginine, L-NA, n = 6 cats; l-nitroarginine methyl ester, L-NAME, n = 5 cats). Both inhibitors caused a decrease in blood flow, basal NO levels, and amplitude of NO fluctuations. There was little change in amplitude for blood flow oscillations, with some enhancement at the lowest frequencies. We conclude that NO is not required for vasomotion and that spontaneous, low-frequency NO fluctuations observed in the cat optic nerve head are a passive phenomenon caused by natural variations in shear stresses.

Incorporating O2-Hb reaction kinetics and the Fåhraeus effect into a microcirculatory O2-CO2 transport model.

The influence of O2-Hb reaction kinetics and the Fåhraeus effect on steady state O2 and CO2 transport in cat brain microcirculation was investigated using our refined multicompartmental model. The most important model predictions include: 1) capillaries are the sites in the microcirculation where the effect of O2-Hb kinetics is most pronounced; 2) while there is only a small difference between equilibrium and actual oxygen saturation, this effect is not negligible; 3) O2-Hb kinetics tends to make the PO2 level at the venous entrance higher than in venules; 4) the influence of the Fåhraeus effect leads to a lower tissue PO2 level than in venules and the outlet vein. The resultant decline in tissue PO2 may lead to a decrease in O2 consumption rate and extraction ratio; 5) although the Fåhraeus effect changes the ratio between arteriolar flux and capillary flux, incorporating the Fåhraeus effect and O2-Hb kinetics into the simulation does not change our previous conclusion, that most of the O2 and CO2 exchange takes place at the capillary level; 6) in general, influences of O2-Hb kinetics and Fåhraeus effect are synergistic; 7) a model that excludes these two mechanisms might overestimate the tissue oxygenation level especially during severe hypoxia.

O2-Hb reaction kinetics and the Fåhraeus effect during stagnant, hypoxic, and anemic supply deficit.

We modified our previous computer model of O2 and CO2 transport in the cerebral microcirculation to include nonequilibrium O2-Hb kinetics and the Fåhraeus effect (reduced tube hematocrit in small microvessels). The model is a steady-state multicompartmental simulation which includes three arteriolar compartments, three venular compartments, and one capillary compartment. Three different types of oxygen deficits (stagnant, hypoxic, and anemic conditions) were simulated by respectively reducing blood flow, arterial O2 saturation, and systemic hematocrit to one half of normal. Microcirculatory distributions for PO2, PCO2, O2 saturation and deviations from equilibrium, and the O2 and CO2 fluxes for each compartment were predicted for the three O2 supply deficits. Differences were found for O2 extraction ratios and relative contributions of arteriolar, venular, and capillary gas fluxes for each type of deficit. The Fåhraeus effect and O2-Hb kinetics reduced O2 extraction in all cases and altered microcirculatory gas distributions depending on the specific type of O2 supply deficits. The modified model continues to predict that capillaries are the major site where gas exchange takes place, and demonstrates that the Fåhraeus effect and nonequilibrium O2-Hb kinetics are important mechanisms that should not be neglected in O2 and CO2 transport modeling. While this model provides useful insight regarding the influence of the Fahraeus effect and O2-Hb kinetics under steady state, the addition of a distributed and dynamic simulation should further elucidate the effects of the brain's heterogeneous properties and transient behavior.

Effect of CO on VO2 of carotid body and chemoreception with and without Ca2+.

This study was done using high PCO (> 500 Torr at PO2 of 120 Torr) in the carotid body perfusate in vitro, and recording simultaneously the activity of the whole carotid sinus nerve (CSN) and VO2 of the carotid body. In the cascade of excitation of CSN by high PCO in the dark [light eliminated the excitation; S. Lahiri, News Physiol. Sci. 9 (1992) 161-165], Ca2+ effects occur at the level of neurosecretion after the level of oxygen consumption, according to the following scheme: CO-hypoxia-->VO2 decrease-->K+ conductance decrease-->cell depolarization-->cytosolic Ca2+ rise-->neurosecretion-->neural discharge. Thus, a part of the hypothesis was that [Ca2+] decrease, being a downstream event, may not affect VO2 of the carotid body. Also, to determine to what extent the intracellular calcium stores contribute to cystolic [Ca2+] and chemosensory discharge with high PCO, we tested the effect of interruption of perfusate flow with medium nominally free of [Ca2+] on CSN excitation and VO2 of the carotid body with and without high PCO. High PCO in the dark decreased carotid body VO2, independent of [Ca2+]o. CSN excitation was always enhanced by high PCO, and its sensitivity to perfusate flow interruption. Also, nominally Ca(2+)-free solution increased the latency and decreased the rate of rise and peak activity of CSN during interruption of perfusate flow, but CO augmented the responses. This reversal effect by CO suggests that Ca2+ is released from intracellular stores, because CO has no other way to excite the chemoreceptors than by acting on the intracellular stores.