A survey of the main technology, biochemical and microbiological features influencing the concentration of biogenic amines of twenty Apulian and Sicilian (Southern Italy) cheeses

Abstract Twenty Apulian and Sicilian cheeses were analysed for their concentrations of eight biogenic amines (BAs), free amino acids, pH, water activity, and subjected to microbiological characterisation. In addition, lactic acid bacteria isolated from cheeses were assayed for their capacity to generate BAs. Principal component analysis was performed to find the effect of different parameters on the distribution of the cheeses. Although short-ripened (≤30 d) cheeses did not show significant BA concentrations, the only BA showing high positive correlation with time of ripening was histamine. Concentration of histidine and, especially, percentage of histidine-decarboxylase bacteria presumably affected histamine concentration. High pH values were negatively correlated to the concentration of tyramine, putrescine, and cadaverine. Fifty percent of the cheeses contained at least one BA at potentially toxic concentrations. Unambiguous and ever-valid relations among parameters and BAs are difficult to determine, because BAs are the result of combined and varied factors

Electrical activity-triggered glucagon-like peptide-1 secretion from primary murine L-cells

Glucagon like peptide 1 (GLP-1) based therapies are now widely used for the treatment of type 2 diabetes. Developing our understanding of intestinal GLP-1 release may facilitate the development of new therapeutics aimed at targeting the GLP-1 producing L-cells. This study was undertaken to characterise the electrical activity of primary L-cells and the importance of voltage gated sodium and calcium channels for GLP-1 secretion. Primary murine L-cells were identified and purified using transgenic mice expressing a fluorescent protein driven by the proglucagon promoter. Fluorescent L-cells were identified within primary colonic cultures for patch clamp recordings. GLP-1 secretion was measured from primary colonic cultures. L-cells purified by flow cytometry were used to measure gene expression by microarray and quantitative RT-PCR. Electrical activity in L-cells was due to large voltage gated sodium currents, inhibition of which by tetrodotoxin reduced both basal and glutamine-stimulated GLP-1 secretion. Voltage gated calcium channels were predominantly of the L-type, Q-type and T-type, by expression analysis, consistent with the finding that GLP-1 release was blocked both by nifedipine and ω-conotoxin MVIIC. We observed large voltage-dependent potassium currents, but only a small chromanol sensitive current that might be attributable to KCNQ1. GLP-1 release from primary L-cells is linked to electrical activity and activation of L-type and Q-type calcium currents. The concept of an electrically excitable L-cell provides a basis for understanding how GLP-1 release may be modulated by nutrient, hormonal and pharmaceutical stimuli

Distinct Potentiation of L-Type Currents and Secretion by cAMP in Rat Chromaffin Cells

We have investigated the potentiating action of cAMP on L-currents of rat chromaffin cells and the corresponding increase of Ca(2+)-evoked secretory responses with the aim of separating the action of cAMP on Ca(2+) entry through L-channels and the downstream effects of cAMP/protein kinase A (PKA) on exocytosis. In ω-toxin-treated rat chromaffin cells, exposure to the permeable cAMP analog 8-(4-chlorophenylthio)-adenosine 3′,5′-monophosphate (pCPT-cAMP; 1 mM, 30 min) caused a moderate increase of Ca(2+) charge carried through L-channels (19% in 10 mM Ca(2+) at +10 mV) and a drastic potentiation of secretion (∼100%), measured as membrane capacitance increments (ΔC). The apparent Ca(2+) dependency of exocytosis increased with pCPT-cAMP and was accompanied by 83% enhancement of the readily releasable pool of vesicles with no significant change of the probability of release, as evaluated with paired-pulse stimulation protocols. pCPT-cAMP effects could be mimicked by stimulation of β(1)-adrenoreceptors and reversed by the PKA inhibitor H89, suggesting strict PKA dependence. For short pulses to +10 mV (100 ms), potentiation of exocytosis by pCPT-cAMP was proportional to the quantity of charge entering the cell and occurred independently of whether L, N, or P/Q channels were blocked, suggesting that cAMP acts as a constant amplification factor for secretion regardless of the channel type carrying Ca(2+). Analysis of statistical variations among depolarization-induced capacitance increments indicates that pCPT-cAMP acts downstream of Ca(2+) entry by almost doubling the mean size of unitary exocytic events, most likely as a consequence of an increased granule-to-granule rather than a granule-to-membrane fusion

Exposure to cAMP and β-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins

T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3–5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca(2+) channels: (1) steep voltage-dependent activation from −50 mV in 10 mm Ca(2+), (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to −30 mV, (4) effective block of Ca(2+) influx with 50 μm Ni(2+), (5) comparable permeability to Ca(2+) and Ba(2+), and (6) insensitivity to common Ca(2+) channel antagonists. The action of exogenous pCPT-cAMP (200 μm) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 μm) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of β-adrenoceptors. Stimulation of β-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of ‘chromaffin cell plasticity’, which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation

Low-Threshold Exocytosis Induced by cAMP-Recruited Ca(V)3.2 (α(1H)) Channels in Rat Chromaffin Cells

We have studied the functional role of Ca(V)3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). Ca(V)3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (−50, −40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 μM Ni(2+), which selectively blocks T-type currents in RCCs. This suggests that the “low-threshold exocytosis” induced by cAMP is due to increased Ca(2+) influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca(2+) entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca(2+) dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited Ca(V)3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca(2+) efficacy similar to that of already existing high-threshold Ca(2+) channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca(2+) current component recruited by cAMP is selectively associated to the α(1H) (Ca(V)3.2) channel isoform

Ca(2+) signaling by T-type Ca(2+) channels in neurons.

Among the major families of voltage-gated Ca(2+) channels, the low-voltage-activated channels formed by the Ca(v)3 subunits, referred to as T-type Ca(2+) channels, have recently gained increased interest in terms of the intracellular Ca(2+) signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca(2+) channels act as an important Ca(2+) source in a wide range of neuronal cell types. The work is focused on T-type Ca(2+) channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca(2+) entering through T-type Ca(2+) channels have been described. Notably, Ca(2+) influx through T-type Ca(2+) channels is the predominant Ca(2+) source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca(2+) signaling through T-type Ca(2+) channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions