A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B 2 bradykinin and M 1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP 3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP 3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. Modulation of ion channels by G protein-coupled receptors is a major mechanism for the regulation of neuronal excitability (1). Many G protein-coupled receptors, including M 1 muscarinic and various peptide receptors, inhibit both the M-type K ϩ current and the N-type Ca 2ϩ current via diffusible second messengers (2-4). Because these receptors typically are linked to phospholipase C (PLC) and the hydrolysis of phosphoinositides (5), it might be supposed that elevations of diacylglycerol, inositol 1,4,5-trisphosphate (IP 3 ), or [Ca 2ϩ ] i underlie the modulation of the M channel and the N-type Ca 2ϩ channel. However, the diffusible messenger(s) for muscarinic action does not appear to be one of those generated by PLC. Evidence for this conclusion in various cells includes: (i) pharmacological activation or block of protein kinase C does not occlude modulation of M current (6, 7); (ii) injection of IP 3 does not always result in suppression of M current (8, 9); and (iii) muscarinic modulation of M current is not accompanied by a measurable [Ca 2ϩ ] i rise in rat sympathetic neurons and occurs even when the cytoplasm contains 20 mM of a Ca 2ϩ buffer (6, 10). Therefore, none of these messengers has been identified as mediating the muscarinic effects (11, 12). Similar conclusions are reached for the modulation of M current by peptides in frog and rat sympathetic neurons (3, 4, 13-15).Bradykinin (BK) recently has been found to depress M current in superior cervical sympathetic ganglion (SCG) neurons by acting on B 2 bradykinin receptors (16). At a supracellular level, it increases norepinephrine outflow from electrically stimulated SCG cultures (17) and raises cardiac contractility when sympathetic neurons are present (18). The suppression of M current in SCG neurons by agonists acting on B 2 bradykinin or M 1 muscarinic receptors is reduced by injection of antibodies against the GTP-binding protein G ␣q͞11 (16,19). Because the search for the second messenger underlying M current inhibition has emphasized the muscarinic pathway, little is known about the signaling pathway activated by B 2 bradykinin receptors in SCG neurons. In many cell types, BK receptors are strongly linked to the G ␣...
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3 untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.Circadian rhythm is a fundamental biological phenomenon in living organisms (10,41,53). To date, efforts to understand the molecular mechanisms of circadian rhythm have focused mainly on transcriptional regulation. A number of studies show that autoregulatory transcriptional-posttranslational feedback loops are crucial for the rhythmic expression of clock-controlled genes (14,30,40,41,46). However, limited data on the posttranscriptional level are available (45). Since mRNA turnover has notable effects on the synthesis of specific proteins and provides the cell with flexibility in achieving rapid changes at the transcript level (9, 35, 50, 52), it is possible that posttranscriptional regulation functions in the rhythmic expression of circadian genes.Recent evidence supports the existence of posttranscriptional mechanisms. In Drosophila, the degradation of Period (per) mRNA modulates its proper circadian fluctuation (49). The accelerated decay of mouse Per1 (mPer1) mRNA in a tau mutant is additionally suggestive of the presence of a posttranscriptional regulatory pathway (32). In transgenic experiments, the differences between the mRNA fluctuations of clock-controlled genes and reporters were tentatively accounted for by variations in their mRNA stability mediated by 3Ј untranslated regions (3ЈUTRs) (22, 51). In computational modeling approaches, mRNA degradation is assumed in the construction of circadian clock models, although its role in rhythm formation is not currently clear (12, 31). Here, we postulate that dynamic mRNA degradation is essential for the formation of circadian rhythms in clock-controlled gene expression, and we support our theory with mathematical modeling and experimental evidence of rat serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) mRNA rhythms.AANAT is a rate-limiting enzyme in the melatonin synthetic pathway that drives the daily rhythm in the leve...
Pancreatic beta-cells are clustered in islets of Langerhans, which are typically a few hundred micrometers in a variety of mammals. In this study, we propose a theoretical model for the growth of pancreatic islets and derive the islet size distribution, based on two recent observations: First, the neogenesis of new islets becomes negligible after some developmental stage. Second, islets grow via a random process, where any cell in an islet proliferates with the same rate regardless of the present size of the islet. Our model predicts either log-normal or Weibull distributions of the islet sizes, depending on whether cells in an islet proliferate coherently or independently. To confirm this, we also measure the islet size by selectively staining islets, which are exposed from exocrine tissues in mice after enzymatic treatment. Indeed revealed are skewed distributions with the peak size of approximately 100 cells, which fit well to the theoretically derived ones. Interestingly, most islets turned out to be bigger than the expected minimal size (approximately 10 or so cells) necessary for stable synchronization of beta-cells through electrical gap-junction coupling. The collaborative behavior among cells is known to facilitate synchronized insulin secretion and tends to saturate beyond the critical (saturation) size of approximately 100 cells. We further probe how the islets change as normal mice grow from young (6 weeks) to adult (5 months) stages. It is found that islets may not grow too large to maintain appropriate ratios between cells of different types. Our results implicate that growing of mouse islets may be regulated by several physical constraints such as the minimal size required for stable cell-to-cell coupling and the upper limit to keep the ratios between cell types. Within the lower and upper limits the observed size distributions of islets can be faithfully regenerated by assuming random and uncoordinated proliferation of each beta-cell at appropriate rates.
Carbon-fiber amperometry detects oxidizable molecules released by exocytosis. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gonadotropes, cultured cerebellar granule cells, and yeast with high concentrations of dopamine (DA) and observed spontaneous and evoked quantal release of DA by amperometry. The rate of detectable spontaneous amperometric events was used as a measure of loading in AtT-20 cells. With 70 mm DA in the bath, loading was complete within 40 min. Cytoplasmic accumulation preceded vesicular loading. Loading decreased proportionally as the bath DA concentration was lowered. Loading rates were similar at 37 and 25 degrees C and much slower at 15 degrees C. Loading was blocked by bafilomycin A(1), a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane DA transporter. Other cells were tested. Spontaneous quantal events became more frequent and evoked events became larger and more frequent when PC12 cells were loaded with DA. Fluid-phase loading of neurons by short stimulation in DA solutions seemed selective for the synaptic vesicles. Thus, many cell types can be loaded with DA to study spontaneous and evoked exocytosis. The amine molecules enter these cells passively and may become concentrated in acidic vesicles by protonation.
A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.
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