Catecholamine exocytosis during low frequency stimulation in mouse adrenal chromaffin cells is primarily asynchronous and controlled by the novel mechanism of Ca2+ syntilla suppression.

Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as 'rest and digest'. How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus-secretion coupling, where exocytosis is synchronized to AP-induced Ca(2+) influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca(2+) influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca(2+) syntillas (i.e. transient, focal Ca(2+) release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca(2+) syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.

Prior Emergency Medical Services Utilization Among People Who Had an Accidental Opioid-Involved Fatal Drug Overdose-Rhode Island, 2018-2020.

OBJECTIVE: To help understand whether decreased emergency medical services (EMS) utilization due to the COVID-19 pandemic contributed to increased accidental fatal drug overdoses, we characterized recent EMS utilization history among people who had an accidental opioid-involved fatal drug overdose in Rhode Island. METHODS: We identified accidental opioid-involved fatal drug overdoses among Rhode Island residents that occurred from January 1, 2018, through December 31, 2020. We linked decedents by name and date of birth to the Rhode Island EMS Information System to obtain EMS utilization history. RESULTS: Among 763 people who had an accidental opioid-involved fatal overdose, 51% had any EMS run and 16% had any opioid overdose-related EMS run in the 2 years before death. Non-Hispanic White decedents were significantly more likely than decedents of other races and ethnicities to have any EMS run (P < .001) and any opioid overdose-related EMS run (P = .05) in the 2 years before death. Despite a 31% increase in fatal overdoses from 2019 through 2020, corresponding with the onset of the COVID-19 pandemic, EMS utilization in the prior 2 years, prior 180 days, or prior 90 days did not vary by time frame of death. CONCLUSION: In Rhode Island, decreased EMS utilization because of the COVID-19 pandemic was not a driving force behind the increase in overdose fatalities observed in 2020. However, with half of people who had an accidental opioid-involved fatal drug overdose having an EMS run in the 2 years before death, emergency care is a potential opportunity to link people to health care and social services.

APP/PS1KI bigenic mice develop early synaptic deficits and hippocampus atrophy.

Abeta accumulation has an important function in the etiology of Alzheimer's disease (AD) with its typical clinical symptoms, like memory impairment and changes in personality. However, the mode of this toxic activity is still a matter of scientific debate. We used the APP/PS1KI mouse model for AD, because it is the only model so far which develops 50% hippocampal CA1 neuron loss at the age of 1 year. Previously, we have shown that this model develops severe learning deficits occurring much earlier at the age of 6 months. This observation prompted us to study the anatomical and cellular basis at this time point in more detail. In the current report, we observed that at 6 months of age there is already a 33% CA1 neuron loss and an 18% atrophy of the hippocampus, together with a drastic reduction of long-term potentiation and disrupted paired pulse facilitation. Interestingly, at 4 months of age, there was no long-term potentiation deficit in CA1. This was accompanied by reduced levels of pre- and post-synaptic markers. We also observed that intraneuronal and total amount of different Abeta peptides including N-modified, fibrillar and oligomeric Abeta species increased and coincided well with CA1 neuron loss. Overall, these data provide the basis for the observed robust working memory deficits in this mouse model for AD at 6 months of age.

Activation of GPCRs modulates quantal size in chromaffin cells through G(betagamma) and PKC.

Exocytosis proceeds by either full fusion or 'kiss-and-run' between vesicle and plasma membrane. Switching between these two modes permits the cell to regulate the kinetics and amount of secretion. Here we show that ATP receptor activation reduces secretion downstream from cytosolic Ca2+ elevation in rat adrenal chromaffin cells. This reduction is mediated by activation of a pertussis toxin-sensitive G(i/o) protein, leading to activation of G(betagamma) subunits, which promote the 'kiss-and-run' mode by reducing the total open time of the fusion pore during a vesicle fusion event. Furthermore, parallel activation of the muscarinic acetylcholine receptor removes the inhibitory effects of ATP on secretion. This is mediated by a G(q) pathway through protein kinase C activation. The inhibitory effects of ATP and its reversal by protein kinase C activation are also shared by opioids and somatostatin. Thus, a variety of G protein pathways exist to modulate Ca2+-evoked secretion at specific steps in fusion pore formation.

Intraneuronal beta-amyloid is a major risk factor--novel evidence from the APP/PS1KI mouse model.

Accumulating evidence points to an important role of intraneuronal beta-amyloid (Abeta) in the development of Alzheimer's disease (AD), with its typical clinical symptoms like memory impairment and changes in personality. We have previously reported on the Abeta precursor protein and presenilin-1 knock-out (APP/PS1KI) mouse model with abundant intraneuronal Abeta(42) accumulation and a 50% loss of CA1 neurons at 10 months of age. In addition, we observed reduced short- and long-term synaptic plasticity, hippocampal neuron loss, and reduced performance in a working memory task. These observations support a pivotal role of intraneuronal Abeta accumulation as a principal pathological trigger in AD.

Preclinical efficacy and safety of KCNH2-G628S gene therapy for postoperative atrial fibrillation.

BACKGROUND: Postoperative atrial fibrillation (POAF) is the most common complication occurring after cardiac surgery. Multiple studies have shown significantly increased risks of stroke, myocardial infarction, and death associated with POAF. Current prophylaxis strategies are inadequate to eliminate this problem. We examined the preclinical efficacy and safety of KCNH2-G628S gene transfer to prevent POAF. METHODS: Domestic pigs received AdKCNH2-G628S by epicardial atrial gene painting and atrial pacemaker implantation for continuous-burst pacing to induce atrial fibrillation. In an initial dose-ranging evaluation, 3 pigs received 5 × 1010 to 5 × 1011 virus particles. In the formal study, 16 pigs were randomized to 3 groups: 5 × 1011 virus particles of AdKCNH2-G628S with 20% Pluronic P407 in saline, 20% Pluronic P407 in saline with no virus, and saline alone. Animals were followed with daily efficacy and safety evaluations through the period of peak adenovirus-mediated transgene expression. After 14 days, pacing was discontinued, and the animals were followed in sinus rhythm for an additional 14 days to assess any longer-term toxicity. RESULTS: In the primary efficacy analysis, the G628S animals exhibited a significant increase in the average time in sinus rhythm compared with the Pluronic control group (59 ± 7% vs 14 ± 6%; P = .009). There was no significant difference between the Pluronic and saline controls (14 ± 6% vs 32 ± 12%; P = .16). Safety assessment showed improved left ventricular function in the G628S animals; otherwise there were no significant differences among the groups in any safety measure. CONCLUSIONS: These data indicate that KCNH2-G628S gene therapy can successfully and safely reduce the risk of AF.

Control of secretion by temporal patterns of action potentials in adrenal chromaffin cells.

Action potentials (APs) are the principal physiological stimuli for neurotransmitter secretion in neurons. Most studies on stimulus-secretion coupling have been performed under voltage clamp using artificial electrical stimuli. To investigate the modulatory effects of AP codes on neural secretion, we introduce a capacitance method to study AP-induced secretion in single cells. The action potential pattern was defined by a four-parameter "code function:" F(n, m, f, d). With this method, cell secretion evoked by stimulation with an AP code was quantified in real time by membrane capacitance (Cm) in adrenal chromaffin cells. We found, in addition to AP frequency (f), for a given number of APs, another parameter of the AP code, the number of AP bursts (m) in which the set of APs occurs, can effectively modulate cell secretion. Possible mechanisms of the m effect are depletion of the readily releasable pool and inactivation of Ca2+ channels during a burst of APs. The physiological m effect may play a key role in AP-mediated neural information transfer within a single neuron and among the elements of a neural network.

Calcium influx through hyperpolarization-activated cation channels (I(h) channels) contributes to activity-evoked neuronal secretion.

The hyperpolarization-activated cation channels (I(h)) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca(2+) can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and I(h) channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca(2+) imaging, we found that there is a Ca(2+) influx in proportion to I(h) induced by hyperpolarization in HEK293 cells. The I(h) channel blockers Cs(+) and ZD7288 inhibit both HCN current and Ca(2+) influx. Measurements of the fractional Ca(2+) current showed that it constitutes 0.60 +/- 0.02% of the net inward current through HCN4 at -120 mV. This fractional current is similar to that of the low Ca(2+)-permeable AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of I(h) for 30 s also resulted in a Ca(2+) influx and an elevated action potential-induced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for I(h) channels in modulating neuronal secretion by permitting Ca(2+) influx at negative membrane potentials.

Na+ channel inactivation: a comparative study between pancreatic islet beta-cells and adrenal chromaffin cells in rat.

A comparative study was carried out on the inactivation of Na+ channels in two types of endocrine cells in rats, beta-cells and adrenal chromaffin cells (ACCs), using patch-clamp techniques. The beta-cells were very sensitive to hyperpolarization; the Na+ currents increased ninefold when the holding potential was shifted from -70 mV to -120 mV. ACCs were not sensitive to hyperpolarization. The half-inactivation voltages were -90 mV (rat beta-cells) and -62 mV (ACCs). The time constant for recovery from inactivation at -70 mV was 10.5 times slower in beta-cells (60 ms) than in ACCs (5.7 ms). The rate of Na+-channel inactivation at physiological resting potential was more than three times slower in beta-cells than in ACCs. Na+ influx through Na+ channels had no effect on the secretory machinery in rat beta-cells. However, these 'silent Na+ channels' could contribute to the generation of action potentials in some conditions, such as when the cell is hyperpolarized. It is concluded that the fractional availability of Na+ channels in beta-cells at a holding potential of -70 mV is about 15 % of that in ACCs. This value in rat beta-cells is larger than that observed in mouse (0 %), but is smaller than those observed in human or dog (90 %).

A novel conotoxin from Conus betulinus, kappa-BtX, unique in cysteine pattern and in function as a specific BK channel modulator.

A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.