Localization of intracellular compartments that exchange Na,K-ATPase molecules with the plasma membrane in a hormone-dependent manner.

BACKGROUND AND PURPOSE: Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. By binding to D1-receptors, dopamine induces endocytosis of plasma membrane Na,K-ATPase, resulting in a reduced capacity of the cells to transport sodium, thus contributing to natriuresis. We have previously demonstrated several aspects of the molecular mechanism by which dopamine induces Na,K-ATPase endocytosis; however, the location of intracellular compartments containing Na,K-ATPase molecules has not been identified. EXPERIMENTAL APPROACH: In this study, we used different approaches to determine the localization of Na,K-ATPase-containing intracellular compartments. By expression of fluorescent-tagged Na,K-ATPase molecules in opossum kidney cells, a cell culture model of proximal tubule epithelia, we used fluorescence microscopy to determine cellular distribution of the fluorescent molecules and the effects of dopamine on this distribution. By labelling cell surface Na,K-ATPase molecules from the cell exterior with either biotin or an epitope-tagged antibody, we determined the localization of the tagged Na,K-ATPase molecules after endocytosis induced by dopamine. KEY RESULTS: In cells expressing fluorescent-tagged Na,K-ATPase molecules, there were intracellular compartments containing Na,K-ATPase molecules. These compartments were in very close proximity to the plasma membrane. Upon treatment of the cells with dopamine, the fluorescence labelling of these compartments was increased. The labelling of these compartments was also observed when the endocytosis of biotin- or antibody-tagged plasma membrane Na,K-ATPase molecules was induced by dopamine. CONCLUSIONS AND IMPLICATIONS: The intracellular compartments containing Na,K-ATPase molecules are located just underneath the plasma membrane.

High-definition mapping of neural activity using voltage-sensitive dyes.

The distribution of patterns of activity in different brain structures has been related to the encoding and processing of sensory information. Consequently, it is important to be able to image the distribution of these patterns to understand basic brain functions. The spatial resolution of voltage-sensitive dye (VSD) methods has recently been enhanced considerably by the use of video imaging techniques. The main factor that now hampers the resolution of VSD patterns is the inherent limitation of the optical systems. Unfortunately, the intrinsic characteristics of VSD images impose important limitations that restrict the use of general deconvolution techniques. To overcomes this problem, in this study an image restoration procedure has been implemented that takes into consideration the limiting characteristics of VSD signals. This technique is based on applying a set of imaging processing steps. First, the signal-to-noise (S/N) ratio of the images was improved to avoid an increase in the noise levels during the deconvolution procedures. For this purpose, a new filter technique was implemented that yielded better results than other methods currently used in optical imaging. Second, focal plane images were deconvolved using a modification of the well-known nearest-neighbor deconvolution algorithm. But to reduce the light exposure of the preparation and simplify image acquisition procedures, adjacent image planes were modeled according to the in-focus image planes and the empirical point spread function (PSF) profiles. Third, resulting focal plane responses were processed to reduce the contribution of optical responses that originate in distant image planes. This method was found to be satisfactory under simulated and real experimental conditions. By comparing the restored and unprocessed images, it was clearly demonstrated that this method can effectively remove the out-of-focus artifacts and produce focal plane images of better quality. Evaluations of the tissue optical properties allowed assessment of the maximum practical optical section thickness using this deconvolution technique in the optical system tested. Determination of the three-dimensional PSF permitted the correct application of deconvolution algorithms and the removal of the contaminating light arising from adjacent as well as distant optical planes. The implementation of this deconvolution approach in salamander olfactory bulb allowed the detailed study of the laminar distribution of voltage-sensitive changes across the bulb layer. It is concluded that (1) this deconvolution procedure is well suited to deconvolved low-contrast images and offers important advantages over other alternatives; (2) this method can be properly used only when the tissue optical properties are first determined; (3) high levels of light scattering in the tissue reduce the optical section capabilities of this technique as well as other deconvolution procedures; and (4) use of the highest numerical aperture in the objectives is advisable because this improves not only the light-collecting efficiency to detect poor-contrast images, but also the spatial frequency differences between adjacent image planes. Under this condition it is possible to overcome some of the limitations imposed by the light scattering/birefringence of the tissue.

Flexible method to obtain high sensitivity, low-cost CCD cameras for video microscopy.

A simple method is described to extend image exposure times in video-rate CCD cameras and thereby, increase their sensitivity and reduce noise level of low-light images. Most commercial video cameras lack the capability of extending image exposures since they operate regular television timing formats. The technique described here implements the control of the exposure times by selectively gating the image readout from the CCD sensor. This prevents the cyclic clearing of photo-charges occurring at regular video-rates, allowing image integration beyond the duration of single video field periods. Image readout is controlled by the duration of external gating pulses, giving the camera an efficient operational versatility under different light conditions. This technique is applicable to standard monochrome and color CCD cameras. The evaluations described here using this technique show that the light sensitivity of an standard video-rate CCD camera can be significantly improved, generating high quality images at low-light levels. These were comparable to those obtained with image intensifiers or intensified video cameras. Cameras are still compatible with regular video equipment, since this technique preserves the normal TV synchronization signals. Results in simulated and real experimental situations confirmed that this technique enables the use of affordable video-rate CCD cameras for a variety of fluorescence microscopy and optical recording applications.

Prolonged effects of short-term fosinopril on blood pressure and vascular morphology and function in rats.

The aim of this study was to evaluate the delayed effects of an angiotensin converting enzyme (ACE) inhibitor on blood pressure and on structural and functional alterations in mesenteric small resistance arteries of spontaneously hypertensive rats (SHR). The ACE inhibitor fosinopril (25 mg/kg/day) was administered according to three different schedules: in one group of SHR from 4 to 8 weeks of age (n = 12), in a second group from 8 to 12 weeks of age (n = 15), and in a third group from 4 to 12 weeks of age (n = 12). Eighteen untreated SHR and 18 untreated Wistar-Kyoto rats served as controls. About half the animals in each group were killed at 13 weeks of age, and the remaining were killed at 38 weeks of age. After death, relative left ventricular mass (left ventricular weight/body weight) was calculated. Vascular morphology (media:lumen ratio) and function (responses to norepinephrine and acetylcholine) in mesenteric small resistance arteries were then assessed using a micromyographic technique. Short-term fosinopril, given either before or after the development of hypertension, persistently reduced (but did not normalize) systolic blood pressure, vascular structural alterations, and reactivity to norepinephrine in mesenteric resistance arteries in SHR. These favorable effects were maintained at least for 26 to 30 weeks after treatment withdrawal. The endothelium-dependent vasodilator response to acetylcholine was improved at 13 but not at 38 weeks of age, in treated SHR. Therefore, the vascular response to norepinephrine seems to be dependent mainly on the structure of the vessels, whereas endothelial function is probably more linked to the hemodynamic load.

Stimulation of protein kinase C rapidly reduces intracellular Na+ concentration via activation of the Na+ pump in OK cells.

Na+ reabsorption is regulated in proximal tubules by hormones that stimulate protein kinase C (PKC). To determine whether stimulation of PKC causes a reduction in intracellular Na+ concentration ([Na+]i) that might link Na+ pump activation to increased Na+ reabsorption, [Na+]i was measured in kidney cells loaded with the Na+-sensitive fluorescent indicator SBFI. Rapid digital imaging fluorescence microscopy determinations were performed in epithelial kidney cells transfected with the rodent Na+ pump alpha1 cDNA. In 42 determinations, the basal [Na+]i was 19.7 +/- 2.4 mM. Stimulation of PKC reduced the [Na+]i to 5.6 +/- 0.6 mM in approximately 10 sec. This drastic change in [Na+]i requires a transient 74-120-fold increase in Na+ pump activity. After the new steady state [Na+]i is reached, the Na+ pump is 58% activated. The entry of Na+ into the cells is not affected by stimulation of PKC; therefore, the reduction in [Na+]i is exclusively dependent on activation of the Na+ pump. Accordingly, PKC stimulation does not affect the [Na+]i of cells expressing a mutant Na+ pump that is not stimulated by PKC. The decrease in [Na+]i observed in cells transfected with the rodent Na+ pump alpha1 cDNA is large and sufficiently fast that it is expected to stimulate rapidly passive Na+-influx into the cells, thereby accounting for the observed PKC-induced stimulation of Na+ reabsorption.

Regulation of Na,K-ATPase transport activity by protein kinase C.

Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase alpha-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the alpha-subunit NH2-terminus is involved in the regulation of Na+, K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase alpha-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the alpha-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent alpha1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type alpha1 cDNA. In contrast, these effects were not observed in cells expressing the NH2-deletion mutant of the alpha-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the alpha-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.

Alpha-adducin gene polymorphism and cardiovascular phenotypes in a general population.

BACKGROUND: Previous studies have shown that molecular variants of the cytoskeletal protein adducin may be involved in regulation of blood pressure both in genetic rat hypertension and in human essential hypertension. OBJECTIVE: To investigate the relationship of genetic polymorphism of alpha-adducin with blood pressure, cardiovascular structure, and some biochemical indexes of cardiovascular risk in a sample of general population. DESIGN AND METHODS: A sample of 246 subjects (124 men and 122 women, aged 57.7+/-3.7 years) was randomly chosen from a middle-aged population. Twenty-four-hour ambulatory blood pressure, as well as left ventricular mass (by echocardiographic methods) and carotid wall thickness (by B-mode ultrasound methods) were measured. DNA was extracted from peripheral blood samples; the Gly460Trp diallelic variant of human alpha-adducin was genotyped by polymerase chain reaction amplification and then allele-specific oligo hybridization. RESULTS: A trend toward higher 24 h ambulatory blood pressure values in subjects not treated with antihypertensive drugs was observed among carriers of Trp460 allele, although the differences did not attain statistical significance (at closest, P = 0.066 for a dominant effect of Trp460 on systolic blood pressure). When blood pressure was considered a dichotomous variable, allowing the inclusion of treated hypertensives), a higher prevalence of Trp460 allele among hypertensives was observed (0.188 versus 0.106 among normotensives, P= 0.02). There was no evidence of association either of left ventricular mass or of common carotid wall thickness with Gly460Trp polymorphism. CONCLUSIONS: In this sample of a general population, the relationship of a genetic polymorphism of alpha-adducin with blood pressure values was rather weak. However, a population-based case-control analysis indicated that there was an association between Trp460 allele and hypertension, with a relative risk for subjects carrying at least one Trp460 allele of approximately 1.6. Further investigation of larger and different population samples in order to assess the role of adducin gene polymorphism as a marker of genetic predisposition to the development of hypertension is warranted.

Angiotensin II type 1 receptor A/C1166 polymorphism. Relationships with blood pressure and cardiovascular structure.

The angiotensin II type 1 (AT1) receptor has a key role in mediating the vasoconstrictor and growth-promoting effects of angiotensin II. It has been reported that a polymorphism of the AT1 receptor gene (an A/C transversion at position 1166) may be associated with cardiovascular phenotypes, such as arterial blood pressure and aortic stiffness, that underlie a condition of increased cardiovascular risk. We examined a sample of 212 subjects randomly selected from a general population in northern Italy to investigate the role of AT1 receptor gene polymorphism, in the regulation of blood pressure and cardiovascular growth. We measured blood pressure (both clinic and 24-hour ambulatory recording), left ventricular mass (echocardiography), and carotid artery wall thickness (B-mode ultrasound); we assessed the AT1 receptor genotype by polymerase chain reaction and allele-specific oligonucleotide hybridization. Blood pressure values were lower in CC homozygotes than in heterozygotes and AA homozygotes; the difference was statistically significant for clinic measurements (mean difference for mean blood pressure, -6.6 mm Hg, P = .01; 95% confidence interval, -1.6 to -11.7 mm Hg) but not for ambulatory blood pressure measurements. CC homozygotes also presented a lower incidence of a positive family history of hypertension (P = .027). No statistically significant differences among AT1 receptor A/C1166 genotypes were observed for left ventricular mass or carotid artery wall thickness. We conclude that the present study does not support a major role of the AT1 receptor gene A/C1166 polymorphism as a marker of conditions associated with increased cardiovascular risk.

Cardiac and vascular structural changes. Prevalence and relation to ambulatory blood pressure in a middle-aged general population in northern Italy: the Vobarno Study.

The aims of this study were to determine the prevalence of structural changes in the carotid arteries and heart and the correlation between these changes and the commonly recognized cardiovascular risk factors in the general population. Structural changes in the carotid arteries were defined as the intima-media thickness of the artery measured by B-mode ultrasound. Changes in the heart were defined as left ventricular mass index (LVMI) measured by echocardiography. LVMI values greater than 134 g/m2 in men and greater than 110 g/m2 in women were considered abnormal, indicating the presence of left ventricular hypertrophy. Blood pressure (BP) was measured in the clinic setting with a mercury sphygmomanometer and by 24-hour noninvasive ambulatory monitoring. Hypertension was defined as a sustained systolic BP greater than or equal to 160 mm Hg and/or diastolic BP increase greater than or equal to 95 mm Hg. The study population consisted of 225 subjects (107 women and 118 men) 48 to 64 years old. Prevalence of intima-media thickening (intima-media thickness > 1 mm) was 11% in normotensive subjects and 44% in hypertensive subjects. The presence of plaque (wall thickening with either mineralization or focal protrusion in the lumen at least 50% greater than the surrounding wall, usually > 2 mm) was observed in 35% of normotensive subjects and 44% of hypertensive subjects. The prevalence of left ventricular hypertrophy was 13% in normotensive subjects and 19% in hypertensive subjects. Intima-media thickness in the common and bifurcation segments of carotid arteries correlated well with LVMI (r = .20 and r = .19, respectively; P < .01). Intima-media thickness and LVMI were both positively related to 24-hour monitored BP (P < .01). However, in the multivariate analysis, body mass index (P = .027), sex (P < .001), and 24-hour mean BP (P = .025) were the most significant determinants of LVMI, whereas carotid artery intima-media thickness was found to be associated best with age (P < .001), cigarette smoking (P = .009), serum cholesterol (P = .025), serum glucose (P = .038), and nighttime systolic BP (P = .006). Logistic regression analysis confirmed the association between the presence of plaque and age (P < .001), nighttime systolic BP (P < .05), and cigarette smoking (P < .05); a negative association between plaque and the decrease in mean systolic BP daytime to nighttime was also observed (P < .001). In conclusion, in a general population of unselected middle-aged subjects, carotid wall thickness and LVMI were associated with each other and related to 24-hour BP levels although the major determinants of carotid wall and cardiac structure were different.

Effects of antihypertensive therapy with angiotensin converting enzyme inhibitors or calcium antagonists on spontaneous cyclic vasomotor activity in small resistance arteries of spontaneously hypertensive rats.

OBJECTIVE: Spontaneous cyclic vasomotor activity can occur in small resistance arteries in vitro after precontraction with a vasoconstrictor. Calcium and potassium channels and nitric oxide synthesis or release seem to be involved in the genesis of this vasomotor activity. We therefore investigated the effects of chronic antihypertensive therapy with calcium antagonists and angiotensin converting enzyme (ACE) inhibitors on the amplitude and frequency of cyclic vasomotor activity in vitro in spontaneously hypertensive rats (SHR). MATERIALS AND METHODS: SHR were treated with fosinopril at 25 mg/kg per day or lacidipine at 10 mg/kg per day or nitrendipine at 30 mg/kg per day, from the age of 4 to 12 weeks. Data were compared with those obtained in untreated SHR and Wistar-Kyoto (WKY) rats. Half the rats were killed at 13 weeks of age, and the remaining half were killed at 38 weeks of age. The mesenteric small resistance arteries were dissected, mounted on a micromyograph and then contracted submaximally with noradrenaline. Acetylcholine was then added to the organ bath. RESULTS: More than 50% of the vessels showed cyclic vasomotor activity. The frequency and amplitude of this activity were greater in SHR than WKY rats after noradrenaline and after acetylcholine. At 13 weeks of age (but not at 38 weeks of age), treatment with a calcium antagonist (either lacidipine or nitrendipine) significantly reduced the frequency and amplitude of the vasomotor activity, probably by interfering with calcium entry. No change was observed after fosinopril. CONCLUSIONS: Antihypertensive treatment with different drugs may affect cyclic vasomotor activity differently, probably by interfering with cellular mechanisms involved in its genesis. The effects of calcium antagonists on cyclic vasomotor activity are still present after short-term but not after long-term treatment withdrawal.

Relationship between sympathetic nervous system activity, baroreflex and cardiovascular effects after acute nitric oxide synthesis inhibition in humans.

OBJECTIVE: To examine the cardiovascular effects of acute systemic nitric oxide synthesis inhibition in humans in relation to the possible involvement of changes in sympathetic nervous system activity or in the baroreceptor reflex. DESIGN: Placebo or NG-monomethyl-L-arginine (250 mg by intravenous infusion for 5 min) was administered to seven healthy male volunteers according to a random, double-blind sequence. METHODS: Blood pressure and heart rate were measured non-invasively using a Finapres device from 20 min before to 80 min after starting infusion; beat-to-beat variability of blood pressure, pulse interval and systolic blood pressure and pulse interval covariation were assessed by means of spectral and sequence analysis methods. Under basal conditions and 15 min and 60 min after infusion, we measured stroke volume and indices of cardiac systolic and diastolic function by echocardiography, forearm blood flow by strain-gauge venous occlusion plethysmography, and plasma catecholamine levels. RESULTS: Compared with placebo, administration of NG-monomethyl-L-arginine caused a transient increase in blood pressure and reduction in heart rate. Stroke volume and indices of cardiac function did not change significantly, whereas cardiac index and forearm blood flow were significantly reduced after 15 min. Spectral analysis of blood pressure and pulse interval showed a significant reduction of power spectral density in the low frequencies (0.03-0.15 Hz) that persisted 60 min after infusion. The plasma noradrenaline level was significantly reduced after 15 min. No change in baroreflex engagement or sensitivity was detected by the cross-spectral or the sequence method. CONCLUSIONS: Acute systemic nitric oxide synthesis inhibition transiently increases blood pressure and reduces heart rate and cardiac index. The acute hypertensive response to NG-monomethyl-L-arginine is dependent neither on sympathetic nervous system activity, which is probably reduced as a consequence of baroreceptor reflex activation, nor on baroreceptor reflex sensitivity, which is not impaired.

Arterial spontaneous rhythmic contractile activity in humans and rats: spectral analysis and regulatory mechanisms.

OBJECTIVE: Many experimental observations have demonstrated the presence of spontaneous cyclic vasomotor activity (CVA) in large and small arteries. This study aimed to evaluate the characteristics of spontaneous CVA in rat and human resistance arteries, and to investigate its possible interference with the evaluation of sympathetic activity by means of spectral analysis of blood pressure in vivo. DESIGN AND RESULTS: In study 1 we examined small mesenteric arteries of spontaneously hypertensive rats and Wistar-Kyoto rats, as well as small omental arteries of normotensive subjects and hypertensive patients (Mulvany and Halpern technique). CVA was enhanced by the agonists of nitric oxide release, and was abolished by the inhibitors of nitric oxide or cyclic GMP synthesis. A potassium channel, which is barium- and zinc-sensitive and tetraethylammonium-insensitive, seems to play a crucial role in the genesis of CVA. In rats and in humans the frequency of CVA fell exactly in the frequency band ('low frequencies') of power spectral analysis of blood pressure usually considered to be an 'index of sympathetic activity'. In study 2, a power spectral analysis of blood pressure variability before and after intra-arterial infusion of noradrenaline or acetylcholine was performed in 18 patients with mild-to-moderate hypertension. The absolute and normalized spectral power of the low-frequency systolic blood pressure peak increased remarkably after noradrenaline and acetylcholine infusion, while its central frequency shifted from 0.10 Hz to approximately 0.06 Hz, exactly the frequency of CVA observed in vitro. CONCLUSIONS: A potassium channel appears to be involved in the genesis of CVA. Also, CVA might contribute to the blood pressure variability independently of the autonomic nervous system activity, and thus probably plays a role in the genesis of the low-frequency peak in the rat and in humans.

Chronic ACE-inhibitor treatment and adrenergic mechanisms in spontaneously hypertensive rats.

We investigated the effects of chronic treatment with the angiotensin-converting enzyme (ACE) inhibitor fosinopril on cardiac and vascular noradrenergic neurotransmission as related to cardiovascular hypertrophy in spontaneously hypertensive rats (SHRs). SHRs were treated with fosinopril at "high dose" (SHR-HD, 25 mg/kg/day) or "low dose" (SHR-LD, 1 mg/kg/day) from the 6th to the 12th week of age, and compared to age-matched untreated SHRs (SHR-C) and Wistar-Kyoto controls (WKY). Blood pressure was significantly reduced in SHR-HD but not in SHR-LD when compared to SHR-C. The antihypertensive dose of fosinopril reduced both cardiac and vascular hypertrophy, whereas the low dose was effective only in reducing vascular hypertrophy. Several differences in presynaptic and postsynaptic cardiovascular noradrenergic neurotransmission were observed between SHR-C and WKY rats (increased cardiac norepinephrine concentration, down-regulation of cardiac beta-adrenoceptors, reduced alpha-adrenergic receptor-mediated vasoconstrictor response of small mesenteric arteries to exogenous norepinephrine). All these differences were abolished by ACE inhibitor treatment, both at antihypertensive or at subantihypertensive doses. The results of this study are consistent with the hypothesis that chronic ACE inhibition may exert an inhibitory modulation on the peripheral adrenergic transmission, which is not related to blood pressure reduction. This modulation does not appear to be a determinant in preventing the development of cardiac hypertrophy but may play a role in the regression of vascular structural alterations in spontaneously hypertensive rats.

Effects of low and high doses of fosinopril on the structure and function of resistance arteries.

It has been suggested that angiotensin-converting enzyme inhibitors may induce a significant regression of cardiovascular hypertrophy not only through blood pressure reduction but also as a possible consequence of growth factor inhibition. The aim of this study was to evaluate the effects of the angiotensin-converting enzyme inhibitor fosinopril, given either at a hypotensive high dose or a nonhypotensive low dose, on structural and functional alterations of mesenteric resistance arteries and on cardiac mass in spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats. Fosinopril was administered in the drinking water from 6 to 12 weeks of age. Rats were killed at 12 weeks, and the ratio of heart weight to body weight was measured. Mesenteric arterioles were dissected and mounted on a micromyograph (Mulvany's technique). Vascular morphology (media-lumen ratio, media thickness) and endothelial function (response to acetylcholine) were then assessed. During the 6 weeks of treatment, systolic pressure in SHR treated with high-dose fosinopril was significantly lower compared with that in untreated SHR, whereas no difference was observed with low-dose fosinopril. In SHR treated with both high-dose and low-dose fosinopril, a statistically significant reduction of vascular structural alterations, in terms of both media-lumen ratio and media thickness, was observed. The ratio of heart weight to body weight was reduced only in SHR treated with high-dose fosinopril. An improvement in the endothelium-dependent relaxation to acetylcholine was observed in SHR treated with high-dose fosinopril compared with untreated SHR, whereas in SHR treated with low-dose fosinopril no improvement in endothelial function was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin-converting enzyme I/D polymorphism and arterial wall thickness in a general population. The Vobarno Study.

BACKGROUND: It has been reported that the D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene is associated with conditions of increased cardiovascular risk, including left ventricular hypertrophy. METHODS AND RESULTS: Considering that a genetically determined overactivity of the renin-angiotensin system may influence cardiac as well as vascular growth, we investigated possible relations between ACE I/D genotype and carotid artery wall thickness (B-mode ultrasound) in 199 subjects, 50 to 64 years old, sampled from the general population of Vobarno, a small town in northern Italy. ACE DD genotype was associated with significantly higher common carotid artery intima-media thickness (P = .003). The occurrence of carotid atherosclerotic plaques was similar in the different genotypes. There was no association of the ACE I/D genotype with blood pressure values (either casual of 24-hour ambulatory monitored). CONCLUSIONS: ACE DD genotype may be considered a risk factor for the development of common carotid intima-media thickening in our study population.

Salamander olfactory bulb neuronal activity observed by video rate, voltage-sensitive dye imaging. II. Spatial and temporal properties of responses evoked by electric stimulation.

1. Video imaging of changes in voltage-sensitive dye (VSD) fluorescence was used to analyze spatial and temporal properties of activity patterns in the in vivo salamander olfactory bulb and primordium piriform cortex after electric stimulation. Distribution of activity among and within the neuronal layers was analyzed after orthodromic stimulation of the whole olfactory nerve (ON), isolated fascicles, or local epithelial sites, and after antidromic stimulation of the medial olfactory tract (OT). 2. Optical signals propagated through the bulbar layers with a sequence that correlates with electrophysiological responses. After orthodromic stimulation, VSD responses started in the glomerular layer, spread to the deeper laminae, and, after reaching the region of mitral/tufted somata, were observed as a brief burst of activity in the OT. Compound action potentials in the ON were associated with short-duration, rapidly depolarizing optical responses in the ON layer. Responses in glomerular layer and external plexiform layer (EPL) first showed in some recordings a brief, small-amplitude hyperpolarization, followed by a period of depolarization, followed by a second, longer-lasting hyperpolarization. The periods of optical hyperpolarization could be related to events observed in intracellular mitral/tufted cell recordings. 3. With shocks delivered to the entire ON, depolarizing responses were nonhomogeneously distributed, appearing as multiple foci or bands of activity. Spatial patterns within each bulbar layer had poorly defined borders. Sites showing short-latency responses were often those with the largest and longest-lasting activity. 4. Increasing the intensity of stimulation to the ON enhanced the size and duration of the depolarizing and hyperpolarizing responses. The short-latency, early hyperpolarization was best seen with low-intensity, peripherally placed stimuli. 5. ON stimulation also elicited activity in the contralateral bulb. Activity started at the innermost layers and spread in patches to regions of the EPL just beneath the glomeruli. These had durations similar to ipsilateral responses, but longer latencies. A period of early hyperpolarization, longer than that on the ipsilateral side, was followed by prolonged depolarization and then by a second, later hyperpolarization. 6. Antidromic stimuli applied to the OT evoked optical responses consisting of a period of depolarization followed by hyperpolarization, similar to the components elicited by orthodromic stimuli. These responses had short time courses, began in the deeper layers, and spread to the superficial region of the bulb usually without reaching the glomerular region. 7. Punctate stimulation of the mucosa or nerve elicited depolarizing and hyperpolarizing events that depended on the stimulation site.(ABSTRACT TRUNCATED AT 400 WORDS)

Salamander olfactory bulb neuronal activity observed by video rate, voltage-sensitive dye imaging. III. Spatial and temporal properties of responses evoked by odorant stimulation.

1. Activity patterns across and within the laminae of the olfactory bulb were analyzed by imaging voltage-sensitive dye responses during odorant stimulation of all or part of the ventral olfactory mucosa. 2. The time course of the signals was generally characterized by a brief, small hyperpolarization, followed by a period of depolarization, and then a longer-lasting hyperpolarization similar to that seen with electric stimulation but with longer durations. 3. The activity was distributed nonhomogeneously across the bulbar laminae in the form of spatially segregated clusters having bandlike appearances. Clusters were observed with three monomolecular odorants, amyl acetate, ethyl-n-butyrate, and limonene, and with the complex odor of meal worms. Although response patterns to different odorants overlapped, they also showed differences in overall distribution. 4. Delivery of high odorant concentrations increased the size of the activated areas and accentuated the degree of response pattern overlap among different odorants. The general properties of the response patterns generated by each odorant were, however, similar at different odorant concentrations and in each of the animals tested. 5. The spatial and temporal distributions of the bulbar responses were somewhat similar regardless of whether the odorants were applied to local epithelial regions via punctate stimulation or to the entire mucosa. Certain regions did, however, have lower thresholds than others for eliciting bulbar activity in response to particular odorants. 6. Odorants applied to regions of the epithelium outside the areas of maximum sensitivity elicited odorant-related activity patterns with depolarizing and hyperpolarizing components similar to those seen with overall stimulation, but only if higher concentrations were used. Activation of distributed odorant sensitivities presumably gave rise to these patterns. 7. These data suggest that subsets of odorant receptor types are found in different areas of the olfactory epithelium, and demonstrate that there is widespread distribution across the epithelium of receptors sensitive to particular odorants. On the basis of the structure of these epithelial fields and the bulb response patterns that they relate to, these findings also provide evidence for complex spatial relationships between the olfactory epithelium and bulb. 8. The findings from this study suggest that representation of odor information in the salamander olfactory bulb does not occur by activation of a few selective bulbar regions, each related to a different odorant species. Instead, large regions of bulbar circuitry are involved in which molecular epitopes may be the unit of representation. Incorporation of new data presented here into a hypothesis of odor coding is discussed.

Salamander olfactory bulb neuronal activity observed by video rate, voltage-sensitive dye imaging. I. Characterization of the recording system.

1. In this paper we describe properties of a video imaging system used to acquire voltage-sensitive dye fluorescence signals from the salamander olfactory bulb. Sources of noise in these signals were evaluated in preparations stained with the potentiometric probe RH-414. These were compared with noise levels in signals obtained from a light-emitting diode array designed to stimulate the experimental conditions with light levels similar to those seen in the salamander bulb recordings. These experiments define a number of determinants of video image quality to standardize optical voltage measurements in the salamander olfactory bulb. 2. Images were acquired at video rates using a Newvicon camera in a standard upright microscope and digitized with an eight-bit video frame grabber. 3. Sources of noise related to camera sensitivity, stability of illumination, and mechanical vibration were characterized. Camera dark noise was less than the pixel variability due to photon noise at the camera faceplate. This pixel noise was the limiting factor for discriminating the spatial and temporal properties of the optical responses. 4. No significant noise was found to be related to image digitization, transmission, or readout by the eight-bit frame grabber. Mechanical vibration, light stability, and other sources of noise could be controlled in vitro. In this condition, voltage-sensitive dye signal noise was similar to that in stimulated experiments using the light-emitting diode array. Higher levels of noise were found in vivo; some of this was reduced by sychronizing frame acquisition to the heartbeat. 5. On the basis of photodiode and video measurements, voltage-sensitive dye responses in the salamander olfactory bulb typically fell between 0.75% and 2.5% fractional change of background fluorescence. By appropriately adjusting the video signals before analog-to-digital conversion, we could detect fractional changes of < 0.5%. 6. Both response averaging and low-bandpass spatial filtering improved the signal-to-noise ratios of the images. For small numbers of averaged runs, the best improvement was obtained by low-bandpass spatial filtering. 7. Acquisition of high-spatial resolution video images permitted the use of low-bandpass spatial filters to suppress pixel noise. The degree of spatial enhancement depended on the relationship between the size of the structures of interest, pixel density, and the properties of the convolution filter kernel. This method avoided exposure of the preparation to prolonged illumination and the necessity of applying the large numbers of repeated stimuli required for averaging.

Are there structural and functional modules in the vertebrate olfactory bulb?

A number of different recording methods have shown that odorants elicit patterns of neuronal activity widely distributed across cells of the olfactory receptor epithelium, olfactory bulb, and piriform cortex in the vertebrate olfactory system. These findings suggest that the physicochemical properties of odorant molecules are processed by distributed coding mechanisms activated in parallel in olfactory circuits in order to characterize a single, "monomolecular" odorant. These findings also suggest that the response patterns seen at higher levels are set up by differential responses in peripheral receptor cells of the olfactory epithelium. One requirement for understanding the details of this proposed encoding scheme is correlation of odor-generated patterns with the components of these circuits. In this paper, results from 2-deoxyglucose and voltage-sensitive dye studies suggest that certain components of these responses may relate to patterns established in reproducibly identifiable aggregates of bulbar cells. These findings are consistent with previous observations suggesting that columnar groups of periglomerular, mitral/tufted and granule cells, oriented perpendicular to the laminae of the bulb, are functionally related to one another. Such cell groups or modules, when activated in parallel, could serve as building block components of the complete ensemble response. According to this hypothesis, different sets of such modules would be activated with different odorant stimuli and modules could be shared to the degree to which the physicochemical properties of the different stimuli overlap.