No abstract

The pentapeptide proctolin is colocalized with a conventional, conductance-increasing neurotransmitter in 3 of 5 excitatory motoneurons that innervate a posture-related tonic flexor muscle of the crayfish. It is released from these neurons in response to nerve impulses. Nanomolar concentrations of proctolin superfused on the tonic flexor muscle act postsynaptically to potentiate tension generated by a given level of depolarization. Proctolin alone has no detectable effect on muscle tension, nor does it alter the resting membrane potential of the muscle. Proctolin produces no detectable effect on the EPSPs of the 1 proctolinergic motoneuron that was examined. Neurally released proctolin can be selectively depleted from severed motor axons following prolonged, low-frequency stimulation; EPSPs reflecting conventional transmitter release are unaltered by this procedure. After proctolin depletion, tension generated by the motoneuron is greatly reduced. Taken together, these results indicate that the peptide secondary transmitter in this neuromuscular preparation is an important contributor to the magnitude of tension generated by the motoneuron, but since its effect is dependent on the depolarizing EPSPs of the conventional neurotransmitter, it does not contribute to the temporal aspects of tension generation. These aspects are controlled exclusively by the conventional neurotransmitter.

The neuropeptide transmitter candidate proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) was associated with three of the five excitatory motoneurons innervating the tonic flexor muscles of the crayfish abdomen. Proctolin immunohistochemical staining occurred in cell bodies and axons of these three identified neurons. Stained axon terminals were detected across the entire tonic flexor muscle. Bioassay of extracts of the tonic flexor muscles indicated the presence of 370 fmol of proctolin/muscle or 670 fmol/mg dry weight. Bioactivity was eliminated in muscles in which the tonic flexor motor root was cut 2 months prior to extraction and in muscle extracts pre-incubated with proctolin antiserum. High pressure liquid chromatography purification of tissue extract indicated that all bioactivity in the crude extract was due to authentic proctolin. Our findings suggest that these three cells function as peptidergic motoneurons. A precedent for this is the proctolin-containing postural motoneuron of the cockroach.

The activity of 2 types of Ca2+ channels (38 and 14 pS in 137 mM Ba2+) in the plasma membrane of the crayfish tonic flexor muscle is modulated by the peptide proctolin. This peptide serves as a cotransmitter in 3 of the 5 excitatory tonic flexor motoneurons and greatly enhances tension after depolarization by the conventional neurotransmitter. Proctolin alone has no effect on these channels, but renders them capable of sustained activity following depolarization. After depolarization induces activity, 5 x 10(-9) M proctolin increases the open probability of the larger channel up to 50-fold due to a marked decrease in the mean channel closed time. There is also at least a 4- fold increase in the percentage of patches with active channels for the large channel and a 2-fold increase for the small channel. Proctolin modulation appears to occur via an intracellular messenger, possibly cAMP. The peptide's effect on channel activity is dose dependent in a manner that parallels its effect on tension. These results indicate that the activation of these channels and the resulting influx of Ca2+ into the muscle fiber play a role in the potentiation of tension in this muscle.

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Clchannel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the F508 mutation (found in ~70% of CF alleles and ~90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous (F508/F508) and compound heterozygous (F508/Other) CF patients, we achieved 28  5 % and 42  15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Clcurrents relative to non-CF controls: 31 5 % in UABCs and 51  3 % in HBECs (both from subjects homozygous for F508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feederfree, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.