Ubaldo García

SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells.

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of γ-aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd2+ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a −50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10−6M [Ca2+]i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from−25 to−10 mV. At 10−9M [Ca2+]i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.

Neurosecretory Role of Crustacean Eyestalk in the Control of Neuronal Activity

From early histological work with methylene blue staining, the existence of neurosecretory cells was postulated in different regions of the eyestalk. Histochemical work rendered similar results (see Gabe, 1966). The most conspicuous system is that composed by the sinus gland, a neurohemal organ located in the distal part of the eyestalk, between the medulla externa and the medulla interna in many species (Fig. 1A). Its basic structure, as seen in Fig. 1B, is that of a bunch of neurosecretory endings, which are the dilated terminals of axons coming from other regions of the eyestalk, to end in apposition to a blood sinus. From morphological and physiological work, the notion was evolved of the sinus gland as the common end of secretory neurons all over the eyestalk and even of incoming fibers from other central ganglia. However, more recently, from experiments with cobalt backfills, a more restricted origin has been advocated, limiting the source of neurosecretory fibers to the sinus gland, to a group of 100–150 cell bodies clustered in the medulla terminalis and known since long ago as the X organ, or Hanstrom’s organ (Andrew et al., 1978; Jaros, 1978). Only a small number of cells outside this cluster were backfilled from the sinus gland.

Inhibition of SERCA pumps induces desynchronized RyR activation in overloaded internal Ca2+ stores in smooth muscle cells

We have previously shown that rapid inhibition of sarcoplasmic reticulum (SR) ATPase (SERCA pumps) decreases the amplitude and rate of rise (synchronization) of caffeine induced-Ca(2+) release without producing a reduction of free luminal SR Ca(2+) level in smooth muscle cells (Gómez-Viquez L, Guerrero-Serna G, García U, Guerrero-Hernández A. Biophys J 85: 370-380, 2003). Our aim was to investigate the role of luminal SR Ca(2+) content in the communication between ryanodine receptors (RyRs) and SERCA pumps. To this end, we studied the effect of SERCA pump inhibition on RyR-mediated Ca(2+) release in smooth muscle cells with overloaded SR Ca(2+) stores. Under this condition, the amplitude of RyR-mediated Ca(2+) release was not affected but the rate of rise was still decreased. In addition, the caffeine-induced Ca(2+)-dependent K(+) outward currents revealed individual events, suggesting that SERCA pump inhibition reduces the coordinated activation of RyRs. Collectively, our results indicate that SERCA pumps facilitate the activation of RyRs by a mechanism that does not involve the regulation of SR Ca(2+) content. Importantly, SERCA pumps and RyRs colocalize in smooth muscle cells, suggesting a possible local communication between these two proteins.

An improved method for long-term measuring of hemolymph fluctuations of non-essential amino acids, GABA and histamine from freely moving crayfish

The microdialysis method was adapted to obtain long-term hemolymph dialysates from the pericardial cavity of freely moving Procambarus clarkii crayfish, to measure fluctuations of non-essential amino acids, GABA and histamine by high-performance liquid chromatography using off-line fluorometric derivatization. Asp, Ala, Tau, GABA and histamine (HA) reached its maximal concentrations at the daybreak, whereas glutamate (Glu), Gln and Gly peaked at the end of the light period. The minimum and maximal detected amounts for each substance along the 24h cycle were (in microM): 20-300Asp, 100-200Glu, 400-700Gln, 400-600Gly, 100-200Tau, 150-300Ala, 2-10 GABA and 25-250HA. Cocktails containing the relative concentration of each amino acid, GABA and histamine resulted in a hyperpolarization that reduced the spontaneous firing of cultured peptidergic X organ neurons. Glu, GABA and histamine evoked a long-lasting hyperpolarization that suppressed the spontaneous firing, whereas Asp, Gly and Tau evoked a depolarization accompanied with neuronal firing. Finally, neither Ala nor Gln modified the resting membrane potential.

Synaptic Regulation of Neurosecretory Cell Activity in the Crayfish Eyestalk

The neurosecretory cells in the crayfish X organ receive an excitatory synaptic input mediated by GABA. GABA can be released from the medulla terminalis in a Ca++-dependent manner. Topical application of GABA and baclofen mimick the synaptically evoked excitation. This is blocked by Picrotoxin. Light induces GABA release from the eyestalk. GABA-like immunoreactive neurones are present in the medulla terminalis