Biomedical subjects

D J Muñoz

Publications and source records attributed to D J Muñoz.

11 recordsLinked to original sources

PubMed2001

Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia.

Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase-luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.02+/-0.04 nmol ATP/mg protein (n = 5), significantly greater (p < 0.001; paired Student's t-test) than the concentration of ATP in the MF (0.36+/-0.05). In the organ of Corti, the VF contained 0.69+/-0.08 nmol ATP/mg protein (n = 4), significantly smaller than the amount in the VF of the lateral wall tissues (p < 0.001; non-paired Student's t-test). Small amounts of fumarase. an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, fluid samples were collected from the first (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% F1O2), ATP concentrations (nM, mean +/- SEM) increased from 6.2+/-2.3 to 9.3+/-4.5 (n = 4), but the difference was not statistically significant. As a result of noise (15 min, 10 kHz, 110 dB SPL. broad band), the ATP levels increased significantly from 7.4+/-1.2 to 16.0+/-1.8 (p = 0.01; Student's t-test: n = 4). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The findings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.

Adenosine Triphosphate↗

PubMed1999

Modulation of cochlear blood flow by extracellular purines.

Humoral adenosine 5'-triphosphate (ATP), adenosine and uridine 5'-triphosphate (UTP) have been shown to have a role in controlling local blood flow in a variety of tissues. The presence of P1 and P2 receptors in the cochlea, and particularly the highly vascular region, the stria vascularis, implies a vasoactive role for these compounds in the inner ear. To test the effect of extracellular purines and pyrimidines on cochlear blood flow, cochleae from anaesthetised guinea-pigs were perfused with ATP (1 microM-10 mM), adenosine (1 microM-10 mM) and UTP (1 mM) in artificial perilymph while blood flow through the cochlea was measured. An acute perilymphatic perfusion technique was established via tubing placed through a hole in the bone overlying scala tympani of the first cochlear turn, with an outlet hole in scala vestibuli of the fourth turn. Blood flow was measured by placing the probe of a laser Doppler blood perfusion monitor on the bone overlying the stria vascularis in the third cochlear turn. ATP and adenosine produced a significant dose dependent increase in cochlear blood flow (28.8-229.0% and 35.8-258.1%, respectively). The effect of ATP (100 microM) on cochlear blood flow was reduced in the presence of reactive blue 2 (1 mM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (1 mM). The blood flow response to adenosine (10 microM) was reduced in the presence of 1,3-dimethylxanthine (theophylline, 100 microM), but not with either 3,7-dimethyl-1-propargylxanthine (10 microM) or 8-cyclopentyl-1,3-dipropylxanthine (10 microM). UTP did not produce any change in the cochlear blood flow. To determine if the ATP effect was also mediated by adenosine derived from ectonucleotidase activity, the perilymphatic compartment was perfused with either ATP plus theophylline (100 microM) or with the non-metabolisable form of ATP, adenosine 5'-O-(3-thiophosphate) (ATPgammaS, 100 microM). The effect of ATP on cochlear blood flow was unaffected with the inclusion of theophylline while ATPgammaS produced an increase in cochlear blood flow similar to the one observed with ATP. These findings indicate that extracellular ATP and its metabolite adenosine have a modulatory role in cochlear blood flow possibly mediated by both P1 and P2 receptors.

Adenosine↗

PubMed1999

P2X receptor-mediated changes in cochlear potentials arising from exogenous adenosine 5'-triphosphate in endolymph.

Our previous studies have determined the presence of adenosine 5'-triphosphate (ATP) in the cochlear fluids and shown that extracellular ATP introduced into the endolymphatic compartment of the guinea pig cochlea has a significant dose-dependent suppressive effect on both endocochlear potential (EP) and cochlear microphonic (CM), which is mediated via P2 receptors. In the present study, the influence of P2 receptor agonists and antagonists on this suppressive effect was investigated to characterise the subtypes of P2 receptor mediating the ATP-induced effect on cochlear function. Using a double-barreled pipette attached to a pressure injector, small volumes (2-10 nl) of ATP (0.01-1 mM) and P2 receptor agonists or P2 receptor antagonists in artificial endolymph were introduced into the scala media of the first (basal) and third turns of the guinea pig cochlea, while the EP and CM were monitored. ATP and P2 receptor agonists (5x10(-14)-1x10(-11)cibacron blue. Neither adenosine nor uridine 5'-triphosphate (2x10(-13)-2x10(-11) moles) nor the P2 receptor antagonists on their own had any effect on EP and CM. The ATP effect on the potentials was greater at the third cochlear turn when compared to the first turn. These results provide evidence that in the endolymphatic compartment of the guinea pig, the extracellular ATP effect on cochlear function is likely mediated through an interaction with P2 receptors which assemble as ATP-gated ion channels.

Adenosine Triphosphate↗

PubMed1998

Ecto-nucleotidases terminate purinergic signalling in the cochlear endolymphatic compartment.

There is strong evidence for a purinergic signalling system in the inner ear which regulates auditory sensitivity. This study describes the terminating mechanism for purinergic signalling in the cochlear endolymphatic compartment via ecto-nucleotidases. Exogenous ATP was introduced into the scala media (SM) of the isolated, perfused guinea-pig cochlea, and the effluent was assayed for the adenine nucleotide metabolites by reverse-phase HPLC. Tissue viability was confirmed by fluorescence imaging of cochlear tissues. Extracellular ATP degradation to adenosine was Ca2+/Mg2+ dependent, and was not affected by inhibitors of intracellular ATPases and non-specific alkaline phosphatase. High azide concentration (5 mM) and suramin produced an inhibitory effect on ATP hydrolysis, consistent with inhibition of E-type ATPase activity. The Vmax of ATP hydrolysis (2564 mumol min-1 SM-1) was indicative of high ecto-ATPase activity. Our results support the role of ecto-nucleotidases as a principal mechanism for termination of purinergic signalling within SM, a compartment of the cochlea showing considerable P2X receptor expression.

Adenosine↗

PubMed1998

The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea.

This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.

5'-Nucleotidase↗

PubMed1996

Ectonucleotidase activity in the perilymphatic compartment of the guinea pig cochlea.

It has been clearly demonstrated that extracellular adenosine 5'-triphosphate (ATP) exerts a potent modulatory activity in the cochlea through its interaction with P2 purinoceptors. However, little is known regarding the metabolism of extracellular ATP in cochlear tissues via ectonucleotidases. This study provides evidence for the presence of ectonucleotidases in the perilymphatic compartment of the guinea pig cochlea. Using microperfusion, ATP (500 microM) was introduced into the cochlear perilymph through the basal turn scala tympani, and effluent was collected from the basal turn scala vestibuli. Samples were subsequently analysed for the presence of adenine metabolites using high performance liquid chromatography (HPLC). Cell viability was evaluated by the activity of the intracellular enzyme lactate dehydrogenase (LDH) in the perfusate. ATP was degraded to 122.8 +/- 9.9 microM (25.0 +/- 5.8%) during the passage through the cochlear perilymphatic compartment. Breakdown of ATP resulted in the formation of adenosine 5'-diphosphate (41.5 +/- 9.0 microM), adenosine 5'-monophosphate (201.3 +/- 15.5 microM), adenosine (108.6 +/- 8.3) and inosine (15.0 +/- 1.5 microM). The degradation of ATP was significantly (P < 0.001, Student's t-test) inhibited in the absence of divalent cations, Ca2+ and Mg2+ in the perfusate. In control experiments, no spontaneous degradation of ATP was observed in vitro. LDH activity was similar during ATP perfusions (2.9 +/- 0.9%) to control perfusions with artificial perilymph (4.2 +/- 1.0%) indicating well preserved cell integrity in the cochlear perilymphatic compartment. The degradation of extracellular ATP in the presence of intact tissues and its inhibition in the absence of divalent cations, a cofactor for ectonucleotidases, provides evidence for ectonucleotidase activity in the perilymphatic fluid space of the cochlea.

Adenine↗

PubMed1995

Extracellular adenosine 5'-triphosphate (ATP) in the endolymphatic compartment influences cochlear function.

There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol; the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.

Adenosine↗

PubMed1995

Adenosine 5'-triphosphate (ATP) concentrations in the endolymph and perilymph of the guinea-pig cochlea.

The concentration of adenosine 5'-triphosphate (ATP) in endolymph (EL), perilymph (PL) and cerebrospinal fluid (CSF), collected from anesthetized guinea pigs was determined using the luciferase-luciferin reaction. The cochlea was exposed by a ventrolateral approach and the bone overlying scala media of the third turn (EL) or scala vestibuli of the first turn (PL) was shaved to a thin layer and a small fenestrum made. For EL sampling, a double-barrelled pipette was inserted through the spiral ligament-stria vascularis complex. One barrel was filled with 150 mM KCl to record the endocochlear potential (EP) and upon the appearance of the positive EP, 0.12-1.22 microliter of fluid was aspirated into the other barrel by gentle negative pressure. For PL sampling, a single-barrelled pipette was advanced into scala vestibuli and 0.3-1.6 microliter of fluid was collected by capillarity. CSF (0.36-1.75 microliter) was obtained from the cisterna magna. The cochleae were removed and processed for light microscopy to determine the extent of tissue damage from the sampling procedure. ATP concentrations (mean +/- SEM, nM) for EL, PL and CSF were 12.95 +/- 2.4 (n = 10), 10.5 +/- 3.9 (n = 11) and 16.1 +/- 5.4 (n = 11) respectively. Differences in ATP concentrations among fluids were not statistically significant. To test the effect of hypoxia on ATP levels, a group of guinea pigs was subjected to a 90 s period of respiratory anoxia prior to sampling of EL, PL or CSF. ATP concentrations were 14.4 +/- 3.5 (n = 11), 20.7 +/- 4.1 (n = 10) and 13.5 +/- 4.6 (n = 4) for EL, PL and CSF, respectively; only PL ATP concentrations were statistically different (P = 0.018, Wilcoxon rank sum test) to basal conditions. This is the first study which demonstrates the presence of free ATP in cochlear fluids. The results indicate that ATP is present in cochlear fluids at concentrations close to those known to cause hair cell depolarization in vitro.

Adenosine Triphosphate↗

PubMed1993

A natriuretic factor is released by saline infusion into rat renal artery.

Saline (154 mM NaCl) infused into one renal artery of anesthetized rats produced a prompt and substantial increase in sodium excretion without significant alteration of arterial pressure, renal blood flow, or glomerular filtration rate. A 1-h infusion at rates of 0.0375, 0.05, or 0.1 ml/min increased sodium excretion by the infused kidney from 0.15 +/- 0.04 to 0.70 +/- 0.12, from 0.45 +/- 0.10 to 2.17 +/- 0.40, and from 0.27 +/- 0.06 to 4.29 +/- 0.80 mumol/min, respectively. Natriuresis also occurred in the contralateral kidney. No comparable responses resulted from infusions at 0.0375 ml/min into the jugular vein, inferior vena cava, or carotid artery. Cross-circulation of blood from infused rats increased sodium excretion in uninfused recipients from 0.36 +/- 0.11 to 1.43 +/- 0.43 mumol.min-1.kidney-1. Isovolemic exchange-transfusion of blood from infused donor rats increased sodium excretion in uninfused recipients from 0.30 +/- 0.05 to 0.51 +/- 0.09 mumol.min-1.kidney-1. In both experiments, aortic infusions below the renal artery produced no comparable effect in recipient rats. Infusion of 300 mM glucose was not natriuretic. Infusion of two NaCl loads, 5.8 and 15.4 mumol/min, each at two different rates, increased sodium excretion in proportion to the load of NaCl and not to the rate of infusion. Infusions of a protein-free artificial rat plasma or of 140 mM NaCl, both with the sodium concentration of rat plasma, produced very little increase in sodium excretion. Infusion of 154 mM sodium lactate produced an increase in sodium excretion comparable to that produced by 154 mM NaCl. Reducing the infusion rate of 154 mM NaCl from 0.05 to 0.01 ml/min produced a rapid decline in the rate of sodium excretion. We conclude that renal arterial saline infusion activates a mechanism that increases sodium excretion by the infused kidney and concurrently causes the release of a natriuretic factor. Increased sodium concentration in renal arterial blood was the only stimulus found to produce the effect.

Animals↗

PubMed1993

Evaluation of renal hormones in natriuresis induced by renal arterial saline infusion.

Low rates of unilateral renal arterial infusion with isotonic saline (154 mM NaCl) produce a natriuresis in both kidneys in anesthetized rats, with the involvement of a blood-borne factor. We investigated whether this response was modulated by known renal or adrenal hormones. The response to saline infusion at 0.05 ml/min was tested after acute adrenalectomy (n = 7), in the presence of various blocking agents [captopril (3 mg.kg-1.h-1, n = 7), indomethacin (5 mg/kg, n = 8), aprotinin (25 KIU/min, n = 8), propranolol (1 micrograms/min, n = 6), or benserazide (15 micrograms/min, n = 6)], and after lignocaine infusion around the renal artery (12.5 micrograms/min, n = 5). In all cases, the overall increase in sodium excretion by both kidneys was not significantly less (alpha < or = 0.01) than that in untreated rats; it was increased by aprotinin, renal nerve blockade, and propranolol. Plasma levels of angiotensin II, aldosterone, and atrial natriuretic peptide were unchanged by renal arterial saline infusion. We conclude that the saline-induced natriuresis is not reduced by inhibition of the production of angiotensin II, prostaglandins, kinins, dopamine, or adrenal hormones, or by factors released by the renal nerves, indicating that none of these is directly responsible for the saline-induced natriuresis.

Adrenalectomy↗

PubMed1992

Effect of Helicobacter pylori infection on colloidal bismuth subcitrate concentration in gastric mucus.

Necropsy gastric mucus infected with Helicobacter pylori has a reduced capacity to concentrate colloidal bismuth subcitrate when compared with non-infected mucus. Mucus mounted in a modified in vitro diffusion chamber was bathed with colloidal bismuth subcitrate solutions at different concentrations and pH levels. Bismuth was measured by atomic absorption spectrophotometry to assess intramucus colloidal bismuth subcitrate concentrations. Bismuth concentrations in non-infected mucus were higher than in Helicobacter pylori infected mucus at all experimental colloidal bismuth subcitrate concentrations and pH levels. Regardless of the infection status, the intramucus concentration of colloidal bismuth subcitrate was dependent upon the concentration of the bathing solution and independent of the pH and the mucus thickness. Colloidal bismuth subcitrate solubility in saline solution varied with pH, and was least soluble in the pH range 1.1 to 3.25 and more soluble above and below this pH range. This study suggests that Helicobacter pylori infection is associated with physicochemical changes in the gastric mucus with a reduction in its capacity to concentrate colloidal bismuth subcitrate. Such a reduction may compromise the attainment of optimum colloidal bismuth subcitrate concentrations necessary for its bactericidal activity.

Adolescent↗