Biomedical subjects

M Díaz-Muñoz

Publications and source records attributed to M Díaz-Muñoz.

At least 19 recordsLinked to original sources

PubMed2005

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells.

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.

Adenosine Triphosphate↗

PubMed2004

Dissociation between adipose tissue signals, behavior and the food-entrained oscillator.

Digestive and metabolic processes are entrained by restricted feeding (RFS) schedules and are thought to be potential elements of a food-entrained oscillator (FEO). Due to the close relationship of leptin with metabolic regulation and because leptin is a relevant communication signal of the individual's peripheral metabolic condition with the central nervous system, we explored whether leptin is an endogenous entraining signal from the periphery to a central element of an FEO. First we characterized in the rat the diurnal rhythm of serum leptin (in rats fed ad libitum (AL)), its adjustment to an RFS and the influence of fasting after RFS, or RFS followed by AL feeding and then total food deprivation (RF-AF) in the persistence of this fluctuating pattern. We also explored the response of free fatty acids and stomach weight under the same entraining conditions. We compared the metabolic response with the behavioral expression of drinking anticipatory activity (AA) under the same conditions. Finally, we tested the effect of daily i.c.v administration of leptin as a putative entraining signal for the generation of AA. Metabolic parameters responded to food entrainment by adjusting their phase to mealtime. However, leptin and free fatty acid rhythms persisted only for a few cycles in fasting conditions and readjusted to the light-darkness cycle after an RF-AF protocol. In contrast, behavioral food-entrained rhythms persisted after both fasting manipulations. Daily leptin i.c.v. administration did not produce AA, nor produce changes in the behavioral free-running rhythm. Stomach weight indicated an adaptive process allowing an extreme stomach distension followed by a slow emptying process, which suggests that the stomach may be playing a relevant role as a storage organ. In conclusion, metabolic signals here studied respond to feeding schedules by adjusting their phase to mealtime, but do only persist for a few cycles in fasting. Leptin does not produce AA and thus is not an entraining signal for FEO. The response of metabolic signals to feeding schedules depends on different mechanisms than the expression of AA.

Adaptation, Physiological↗

PubMed2003

Liver 5'-deiodinase activity is modified in rats under restricted feeding schedules: evidence for post-translational regulation.

Restricted feeding schedules (RFSs) produce a behavioral activation known as anticipatory activity, which is a manifestation of a food-entrained oscillator (FEO). The liver could be playing a role in the physiology of FEO. Here we demonstrate that the activity of liver selenoenzyme deiodinase type 1 (D1), which transforms thyroxine into triiodothyronine (T3), decreases before food access and increases after food presentation in RFSs. These changes in D1 activity were not due to variations in D1 mRNA. In contrast, a 24 h fast promoted a decrease in both D1 activity and mRNA content. The adjustment in hepatic D1 activity was accompanied by a similar modification in T3-dependent malic enzyme, suggesting that the local generation of T3 has physiological implications in the liver. These results support the notion that the physiological state of rats under RFSs is unique and distinct from rats fed freely or fasted for 24 h. Data also suggest a possible role of hepatic D1 enzyme in coordinating the homeorhetic state of the liver when this organ participates in FEO expression.

Animals↗

PubMed2001

A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases.

A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.

Amino Acid Sequence↗

PubMed2001

Adenosine reverses a preestablished CCl4-induced micronodular cirrhosis through enhancing collagenolytic activity and stimulating hepatocyte cell proliferation in rats.

Cirrhosis is one of the most common causes of mortality worldwide, because hepatic dysfunction constitutes a potentially lethal condition. Having demonstrated the hepatoprotective effect of adenosine against CCl(4)-induced cirrhosis, the present study was aimed at assessing adenosine's effect on an already-established micronodular cirrhosis. Chronic administration of CCl(4) (10 weeks) induced a cirrhotic state, characterized by increased liver fibronectin and collagen types I and III content, enhanced expression of alpha-1 (I) collagen mRNA, portal hypertension, and liver dysfunction. After CCl(4) discontinuation (5 weeks), increased persitance of alpha-1 (I) collagen mRNA expression and deposition, enhanced proline incorporation into collagen and prolyl hydroxylase activity evidenced active fibrogenesis. Several weeks after CCl(4) withdrawal, deposited collagen showed an enhanced type I/III ratio, which was associated with deficient collagenolytic activity in cirrhotic livers. Liver expression of some metalloproteinases (MMPs) and of tissue inhibitors of MMPs (TIMPs) also indicated decreased collagen breakdown in cirrhotic livers. Parameters indicative of oxidative stress (mainly protein oxidation) were persistently augmented. These events were coincident with diminished regenerative capacity of the cirrhotic liver. Intraperitoneal adenosine administration to CCl(4)-induced cirrhotic rats blocked active fibrogenesis and increased the collagen degradation (most probably by decreasing liver TIMPs levels), normalizing collagen-type ratios. In addition, the nucleoside promoted an effective hepatocyte's proliferation in the cirrhotic liver and accelerated normalization of parameters indicative of liver function and oxidative stress. Thus, adenosine readily reversed an experimental cirrhosis through stimulating liver collagenolytic and proliferative capacities, as well as by accelerating functional recovery.

Adenosine↗

PubMed2001

Functional expression of recombinant type 1 ryanodine receptor in insect cells.

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.

Animals↗

PubMed2000

Anticipatory changes in liver metabolism and entrainment of insulin, glucagon, and corticosterone in food-restricted rats.

Restricted feeding schedules entrain behavioral and physiological circadian rhythms, which depend on a food-entrainable oscillator (FEO). The mechanism of the FEO might depend on digestive and endocrine processes regulating energy balance. The present study characterizes the dynamics of circulating corticosterone, insulin, and glucagon and regulatory parameters of liver metabolism in rats under restricted feeding schedules. With respect to ad libitum controls, food-restricted rats showed 1) an increase in corticosterone and glucagon and a decrease in insulin before food access, indicating a predominant catabolic state; and 2) a reduction in lactate-to-pyruvate and beta-hydroxybutyrate-to-acetoacetate ratios, indicating an oxidized cytoplasmic and mitochondrial redox state in the liver metabolism. All these changes were reversed after feeding. Moreover, liver energy charge in food-restricted rats did not show a significant modification before feeding, despite an increase in adenine nucleotides, but showed an important decrease after food intake. Variations detected in the liver of food-restricted rats are different from those prevailing under 24-h fasting. These observations suggest "anticipatory activity" of the liver metabolism to optimize the processing of nutrients to daily feeding. Data also suggest a possible relationship of the liver and endocrine signals with the FEO.

3-Hydroxybutyric Acid↗

PubMed1999

Redox state and energy metabolism during liver regeneration: alterations produced by acute ethanol administration.

Ethanol metabolism can induce modifications in liver metabolic pathways that are tightly regulated through the availability of cellular energy and through the redox state. Since partial hepatectomy (PH)-induced liver proliferation requires an oversupply of energy for enhanced syntheses of DNA and proteins, the present study was aimed at evaluating the effect of acute ethanol administration on the PH-induced changes in cellular redox and energy potentials. Ethanol (5 g/kg body weight) was administered to control rats and to two-thirds hepatectomized rats. Quantitation of the liver content of lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, and adenine nucleotides led us to estimate the cytosolic and mitochondrial redox potentials and energy parameters. Specific activities in the liver of alcohol-metabolizing enzymes also were measured in these animals. Liver regeneration had no effect on cellular energy availability, but induced a more reduced cytosolic redox state accompanied by an oxidized mitochondrial redox state during the first 48 hr of treatment; the redox state normalized thereafter. Administration of ethanol did not modify energy parameters in PH rats, but this hepatotoxin readily blocked the PH-induced changes in the cellular redox state. In addition, proliferating liver promoted decreases in the activity of alcohol dehydrogenase (ADH) and of cytochrome P4502E1 (CYP2E1); ethanol treatment prevented the PH-induced diminution of ADH activity. In summary, our data suggest that ethanol could minimize the PH-promoted metabolic adjustments mediated by redox reactions, probably leading to an ineffective preparatory event that culminates in compensatory liver growth after PH in the rat.

Animals↗

PubMed1999

Circadian modulation of the ryanodine receptor type 2 in the SCN of rodents.

We examined the temporal modulation of intracellular calcium release channels in the suprachiasmatic nucleus (SCN). We found a circadian rhythm in [3H]ryanodine binding that was specific to the SCN. The peak in the rhythm occurred at CT 7 and was due to an increase in Bmax, which correlated well with immunoblots showing an increase in RyR-2 expression in the SCN. Double immunohistochemical studies showed that RyR-2 was expressed exclusively in neurons. Ryanodine and caffeine applied around CT 7-9 advanced the clock phase in a hamster brain slice preparation. No rhythm of IP3R was seen in any of the brain areas studied. Our results indicate that RyR-2 exhibits an endogenous rhythm, which influences the intracellular calcium dynamics and thus modulates SCN activity.

Animals↗

PubMed1999

Potentiation of prolactin secretion following lactotrope escape from dopamine action. II. Phosphorylation of the alpha(1) subunit of L-type, voltage-dependent calcium channels.

Modulation of Ca(2+) channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca(2+) channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca(2+) currents in GH(4)C(1) lactotropic clonal cells transfected with a cDNA encoding the long form of the human D(2)-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH(4)C(1)/D(2)-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[(3)H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [(3)H]azidopine revealed a protein consistent with the alpha(1) subunit of L-type Ca(2+) channels of 165-170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative alpha(1) subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the alpha(1C) and alpha(1D) subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the alpha(1) subunit of high voltage activated, L-type Ca(2+) channels is responsible for the facilitation of Ca(2+) currents in lactotropes, and hence for the potentiation of secretagogue-mediated PRL secretion. Thus, phosphorylation of Ca(2+) channels in endocrine cells may be a mechanism for the regulation of various functions including amplification of hormone secretion.

Animals↗

PubMed1999

Correlation between blood adenosine metabolism and sleep in humans.

Blood adenosine metabolism, including metabolites and metabolizing enzymes, was studied during the sleep period in human volunteers. Searching for significant correlations among biochemical parameters found: adenosine with state 1 of slow-wave sleep (SWS); activity of 5'-nucleotidase with state 2 of SWS; inosine and AMP with state 3-4 of SWS; and activity of 5'-nucleotidase and lactate with REM sleep. The correlations were detected in all of the subjects that presented normal hypnograms, but not in those who had fragmented sleep the night of the experiment. The data demonstrate that it is possible to obtain information of complex brain operations such as sleep by measuring biochemical parameters in blood. The results strengthen the notion of a role played by adenosine, its metabolites and metabolizing enzymes, during each of the stages that constitute the sleep process in humans.

Adenosine↗

PubMed1998

Modifications of intracellular calcium release channels and calcium mobilization following 70% hepatectomy.

The aim of this study was to investigate the properties of ryanodine and IP3 receptors in regenerating liver following 70% hepatectomy, and to evaluate the hepatic Ca2+ distribution and mobilization during this process. Specific [3H]ryanodine and [3H]IP3 binding to hepatic smooth endoplasmic reticulum membranes, as well as subcellular Ca2+ determination by atomic absorption flame photometry and Ca2+ mobilization by INDO-1 AM spectrofluorescence in hepatocytes, was performed in regenerating livers after surgical 70% hepatectomy. Incorporation of 14C amino acids into proteins and of 32P into phospholipids was done in subcellular fractions. Ryanodine receptor Kd presented a dramatic increase after 12 h of surgery and remained high up to 2 days of treatment. IP3 receptor Bmax showed a significant augmentation starting at 6 h after hepatectomy and returning to normal values after 1 week. Cytosolic total calcium content decreased from 12 h until 4 days after hepatectomy whereas the microsomal and mitochondrial total calcium increased at 1 and 2-4 days of liver regeneration, which coincided with the differential turnover of proteins and phospholipids in these fractions. ATP-induced Ca2+ transients in hepatocytes of 24-h-hepatectomized rats confirmed the altered sensitivity of the ryanodine receptor toward its ligand, since 10 times more ryanodine was necessary to alter the ATP-induced Ca2+ transient. The data support the notion that the calcium release channels are targets of mechanisms of metabolic control during the proliferative response following 70% hepatectomy and might be part of the modified intracellular Ca2+ dynamics during liver regeneration.

Animals↗

PubMed1998

Persistence of metabolic rhythmicity during fasting and its entrainment by restricted feeding schedules in rats.

The presence of a food-entrainable oscillator (FEO) independent from the SCN is now well established, but until now its location and characterization have been elusive. Because its expression requires priming of the animal's metabolism toward a catabolic state, it is possible that metabolic rhythms may be related to FEO. The present study was designed to determine whether metabolic rhythms persist during fasting and whether such rhythms could be entrained to a restricted feeding schedule. The results indicate persistent rhythms of triacylglycerides, free fatty acids, glucose, and proteins during fasting, whereas ketone bodies and liver glycogen changed their concentration as a function of fasting. Daily food pulses of 2 h entrained the rhythms of triacylglycerides and free fatty acids and restored ketone bodies and liver glycogen to similar levels as controls. Neither glucose nor proteins were affected by the food pulse. These results indicate the relevance of lipid metabolism as a phenomenon associated with the FEO.

Animals↗

PubMed1998

Decrease in muscarinic M2 receptors from synaptosomes in the pons and hippocampus after REM sleep deprivation in rats.

The effects of both REM sleep deprivation and its recovery on pontine and hippocampus muscarinic M2 receptors were investigated in synaptosomes using [3H]-AF-DX 384 as a ligand. Animals were divided into three groups: REM sleep deprivation group (small platforms 6.5 cm of diameter); stress group (large platforms 14 cm of diameter) and cage control group. In a second experiment REM sleep-deprived animals were allowed 48 h of recovery. REM sleep-deprived rats showed a reduction in M2 receptors compared with both intact and stress groups. Changes in M2 receptors were also observed after 48 h of recovery from REM sleep deprivation only in hippocampus. The enhancement of acetylcholine release during both REM sleep deprivation and recovery could explain the present findings.

Animals↗

PubMed1997

Balance between oxidative damage and proliferative potential in an experimental rat model of CCl4-induced cirrhosis: protective role of adenosine administration.

Oxidative stress and its consequent lipid peroxidation (LP) exert harmful effects, which have been currently involved in the generation of carbon tetrachloride-induced cirrhosis. However, the recent report that "physiological" LP can be associated with liver regeneration (LR) makes it necessary to discriminate between oxidative stress-induced and LR-associated LP. In rats rendered cirrhotic by continuous CCl4 administration for 4 weeks, moderate cell necrosis and fine fatty infiltration were found. The histological abnormalities were accompanied by increased LP, mainly accounted for by the microsomal and cytosolic fractions and evidence of oxidative stress (decreased hepatic glutathione content and changes in xanthine oxidase and pentose phosphate pathway activities). After 8 weeks, a micronodular cirrhosis developed, but oxidative stress was greatly attenuated, only persisting in the enhanced LP confined to microsomes. Simultaneous administration of adenosine, a reliable hepatoprotector that readily prevents the onset of liver fibrosis, was able to block the oxidative stress induced by the long-term CCl4 treatment but elicited a selective subcellular distribution of increased LP, similar to that found during LR. The adenosine-induced changes in liver LP (mainly in the nuclear fraction) correlated with an increased activity of thymidine kinase. Therefore, data suggest that adenosine-mediated preservation of energy availability and mitochondrial function could participate in preventing the onset of oxidative stress in cirrhotic rats. The latter could induce a successful liver recovery, curtailing the sequence of events leading to fibrogenesis.

Adenosine↗

PubMed1997

Effects of N omega-nitro-L-arginine and L-arginine on quinolinic acid-induced lipid peroxidation.

The effects of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NARG), and a nitric oxide precursor, L-arginine (L-ARG), on the lipid peroxidation induced by quinolinic acid (QUIN, an NMDA receptor agonist), were both tested in synaptosomal fractions from whole rat brain. Baseline of lipid peroxidation was found at 2.43 +/- 0.24 fluorescence units/mg protein or 14.27 +/- 1.24 nmoles of TBARS/mg protein (100%). QUIN (100 microM)-induced lipid peroxidation in synaptosomes (256% and 166% vs. control, as measured by lipid fluorescent products and thiobarbituric acid-reactive substances, respectively) was inhibited by concentrations of 10, 40, 100, 200 and 400 microM of L-NARG (74%, 58%, 56%, 48% and 48% vs. quinolinate value, respectively). Coincubation of synaptosomes with QUIN plus L-ARG (100 microM), which alone resulted a potent pro-oxidant (277% vs. control), increased the lipoperoxidative effect induced by QUIN alone in 120% (290% vs. control). Synaptosomes simultaneously exposed to QUIN (100 microM) plus L-ARG (100 microM) plus L-NARG (200 microM) showed levels of lipid peroxidation similar to those of quinolinate alone. These findings suggest that nitric oxide may contribute to the oxidative damage induced in vitro by QUIN.

Animals↗

PubMed1996

Temporal variations of adenosine metabolism in human blood.

Eight diurnally active (06:00-23:00 h) subjects were adapted for 2 days to the room conditions where the experiments were performed. Blood sampling for adenosine metabolites and metabolizing enzymes was done hourly during the activity span and every 30 min during sleep. The results showed that adenosine and its catabolites (inosine, hypoxanthine, and uric acid), adenosine synthesizing (S-adenosylhomocysteine hydrolase and 5'-nucleotidase), degrading (adenosine deaminase) and nucleotide-forming (adenosine kinase) enzymes as well as adenine nucleotides (AMP, ADP, and ATP) undergo statistically significant fluctuations (ANOVA) during the 24 h. However, energy charge was invariable. Glucose and lactate chronograms were determined as metabolic indicators. The same data analyzed by the chi-square periodogram and Fourier series indicated ultradian oscillatory periods for all the metabolites and enzymatic activities determined, and 24-h oscillatory components for inosine, hypoxanthine, adenine nucleotides, glucose, and the activities of SAH-hydrolase, 5'-nucleotidase, and adenosine kinase. The single cosinor method showed significant oscillatory components exclusively for lactate. As a whole, these results suggest that adenosine metabolism may play a role as a biological oscillator coordinating and/or modulating the energy homeostasis and physiological status of erythrocytes in vivo and could be an important factor in the distribution of purine rings for the rest of the organism.

5'-Nucleotidase↗

PubMed1995

Properties of the ryanodine-sensitive release channels that underlie caffeine-induced Ca2+ mobilization from intracellular stores in mammalian sympathetic neurons.

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca2+ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [3H]ryanodine binding and recording of Ca2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca2+ measurements showed that these cells possess caffeine-sensitive intracellular Ca2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca2+ showed a complex dependence on [Ca2+]i. Transient elevations of [Ca2+]i by 50-500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]i reached higher levels. The caffeine-induced Ca2+ release and its dependence on [Ca2+]i was further examined by [3H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (KD = 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax = 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [3H]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1-10 microM that was further increased by caffeine. Caffeine-dependent enhancement of [3H]ryanodine binding increased and then decreased as [Ca2+] rose, with an optimum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 and 10 microM. At 100 microM [Ca2+], caffeine and ATP enhanced [3H]ryanodine binding by 35 and 170% respectively, while binding was reduced by > 90% with ruthenium red and MgCl2. High-conductance (240 pS) Ca2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca2+ from the cytosolic side, and were blocked by Mg2+ and ruthenium red. Ryanodine (2 microM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca2+ release. Our data also suggest that the release of Ca2+ by caffeine has a bell-shaped dependence on Ca2+ in the physiological range of cytoplasmic [Ca2+].

Adrenergic Fibers↗